Chemically modified proteins with a carbohydrate moiety

ABSTRACT

The present invention relates to a chemically modified mutant protein including a cysteine residue substituted for a residue other than cysteine in a precursor protein, the substituted cysteine residue being subsequently modified by reacting the cysteine residue with a glycosylated thiosulfonate. Also, a method of producing the chemically modified mutant protein is provided. The present invention also relates to a glycosylated methanethiosulfonate. Another aspect of the present invention is a method of modifying the functional characteristics of a protein including providing a protein and reacting the protein with a glycosylated methanethiosulfonate reagent under conditions effective to produce a glycoprotein with altered functional characteristics as compared to the protein. In addition, the present invention relates to methods of determining the structure-function relationships of chemically modified mutant proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional PatentApplication Serial No. 60/091,687, filed Jul. 2, 1998, and U.S.Provisional Patent Application Serial No. 60/131,446, filed Apr. 28,1999, and which are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to chemically modifies mutantproteins having modified glycosylation patterns with respect to aprecursor protein from which they are derived. In particular, thepresent invention relates to a chemically modified mutant proteinincluding a cysteine residue substituted for a residue other thancysteine in a precursor protein, the substituted cysteine residue beingsubsequently modified by reacting the cysteine residue with aglycosylated thiosulfonate. The present invention also relates to amethod of producing the chemically modified mutant proteins and aglycosylated methanethiosulfonate. Another aspect of the presentinvention is a method of modifying the functional characteristics of aprotein by reacting the protein with a glycosylated methanethiosulfonatereagent. The present invention also relates to methods of determiningthe structure-function relationships of chemically modified mutantproteins.

BACKGROUND OF THE INVENTION

[0003] Modifying enzyme properties by site-directed mutagenesis has beenlimited to natural amino acid replacements, although molecularbiological strategies for overcoming this restriction have recently beenderived (Cornish et al., Angew. Chem., Int. Ed. Engl., 34:621-633(1995)). However, the latter procedures are difficult to apply in mostlaboratories. In contrast, controlled chemical modification of enzymesoffers broad potential for facile and flexible modification of enzymestructure, thereby opening up extensive possibilities for controlledtailoring of enzyme specificity.

[0004] Changing enzyme properties by chemical modification has beenexplored previously, with the first report being in 1966 by the groupsof Bender (Polgar et al., J. Am. Chem. Soc., 88:315-3154 (1966)) andKoshland (Neet et al., Proc. Natl. Acad. Sci. USA, 56:1606-1611 (1966)),who created a thiolsubtilisin by chemical transformation (CH₂OH→CH₂SH)of the active site serine residue of subtilisin BPN′ to cysteine.Interest in chemically produced artificial enzymes, including some withsynthetic potential, was renewed by Wu (Wu et al., J. Am. Chem. Soc.111:4514-4515 (1989); Bell et al., Biochemistry, 32:3754-3762 (1993))and Peterson (Peterson et al., Biochemistry, 34:6616-6620 (1995)), and,more recently, Suckling (Suckling et al., Bioorg. Med. Chem. Lett.,3:531-534 (1993)).

[0005] Enzymes are now widely accepted as useful catalysts in organicsynthesis. However, natural, wild-type, enzymes can never hope to acceptall structures of synthetic chemical interest, nor always be transformedstereospecifically into the desired enantiomerically pure materialsneeded for synthesis. This potential limitation on the syntheticapplicabilities of enzymes has been recognized, and some progress hasbeen made in altering their specificities in a controlled manner usingthe site-directed and random mutagenesis techniques of proteinengineering. However, modifying enzyme properties by protein engineeringis limited to making natural amino acid replacements, and molecularbiological methods devised to overcome this restriction are not readilyamenable to routine application or large scale synthesis. The generationof new specificities or activities obtained by chemical modification ofenzymes has intrigued chemists for many years and continues to do so.

[0006] U.S. Pat. No. 5,208,158 to Bech et al. (“Bech”) describeschemically modified detergent enzymes where one or more methionines havebeen mutated into cysteines. The cysteines are subsequently modified inorder to confer upon the enzyme improved stability towards oxidativeagents. The claimed chemical modification is the replacement of thethiol hydrogen with C₁₋₆ alkyl.

[0007] Although Bech has described altering the oxidative stability ofan enzyme through mutagenesis and chemical modification, it would alsobe desirable to develop one or more enzymes with altered properties suchas activity, nucleophile specificity, substrate specificity,stereoselectivity, thermal stability, pH activity profile, and surfacebinding properties for use in, for example, deterrents or organicsynthesis. In particular, enzymes, such as subtilisins, tailored forpeptide synthesis would be desirable. Enzymes useful for peptidesynthesis have high esterase and low amidase activities. Generally,subtilisins do not meet these requirements and the improvement of theesterase to amidase selectivities of subtilisins would be desirable.However, previous attempts to tailor enzymes for peptide synthesis bylowering amidase activity have generally resulted in dramatic decreasesin both esterase and amidase activities. Previous strategies forlowering the amidase activity include the use of water-miscible organicsolvents (Barbas et al., J. Am. Chem. Soc., 110:5162-5166 (1988); Wonget al., J. Am. Chem. Soc., 112:945-953 (1990); and Sears et al.,Biotechnol. Prod., 12:423-433 (1996)) and site-directed mutagenesis(Abrahamsen et al., Biochemistry, 30:4151-4159 (1991); Bonneau et al.,“Alteration of the Specificity of Subtilisin BPN′ by Site-DirectedMutagenesis in its S1 and S1′ Binding-Sites,” J. Am. Chem. Soc.,113:1026-1030 (1991); and Graycar et al., Ann. N.Y. Acad. Sci., 67:71-79(1992)). However, while the ratios of esterase-to-amidase activitieswere improved by these approaches, the absolute esterase activities werelowered concomitantly. Abrahamsen et al., Biochemistry, 30:4151-4159(1991). Chemical modification techniques (Neet et al., Proc. Nat. Acad.Sci., 56:1606 (1966); Polgar et al., J. Am. Chem. Soc., 88:3153-3154(1966); Wu et al., J. Am. Chem. Soc., 111:4514-4515 (1989); and West etal., J. Am. Chem. Soc., 112:5313-5320 (1990)), which permit theincorporation of unnatural amino acid moieties, have also been appliedto improve esterase to amidase selectivity of subtilisins. For example,chemical conversion of the catalytic triad serine (Ser221) of subtilisinto cysteine (Neet et al., Proc. Nat. Acad. Sci., 56:1606 (1966); Polgaret al., J. Am. Chem. Soc., 88:3153-3154 (1966); and Nakatsuka et al., J.Am. Chem. Soc., 109:3808-3810 (1987)) or to selenocysteine (Wu et al.,J. Am. Chem. Soc., 111:4514-4515 (1989)), and methylation of thecatalytic triad histidine (His57) of chymotrypsin (West et al., J. Am.Chem. Soc., 112:5313-5320 (1990)), effected substantial improvement inesterase-to-amidase selectivities. Unfortunately however, thesemodifications were again accompanied by 50- to 1000-fold decreases inabsolute esterase activity.

[0008] Surface glycoproteins act as markers in cell-cell communicationevents that determine microbial virulence (Sharon et al., EssaysBiochem., 30:59-75 (1995)), inflammation (Lasky, Annu. Rev. Biochem.,64:113-139 (1995); Weis et al., Annu. Rev. Biochem., 65:441-473 (1996)),and host immune responses (Varki, Glycobiol., 3:97-130 (1993); Dwek,Chem. Rev., 96:683-720 (1996)). In addition. the correct glycosylationof proteins is critical to their expression and folding (Helenius, Mol.Biol. Cell, 5:253-265 (1994)) and increases their thermal andproteolytic stability (Opdenakker et al., FASEB J., 7:11330-1337(1993)). Glycoproteins occur naturally in a number of forms (glycoforms)(Rademacher et al., Annu. Rev. Biochem., 57:785-838 (1988)) that possessthe same peptide backbone, but differ in both the nature and site ofglycosylation. The differences exhibited (Rademacher et al., Annu. Rev.Biochem., 57:785-838 (1988); Parekh et al., Biochem., 28:7670-7679(1989); Knight, Biotechnol., 7:35-40 (1989)) by each component withinthese microheterogeneous mixtures present regulatory difficulties (Liu,Trends Biotechnol., 10:114-120 (1992); Bill et al., Chem. Biol.,3:145-149 (1996)) and problems in determining exact function. To explorethese key properties, there is a pressing need for methods that will notonly allow the preparation of pure glycosylated proteins, but will alsoallow the preparation of non-natural variants for the determination ofstructure-function relationships, such as structure-activityrelationships (SARs). The few studies that have compared singleglycoforms successfully have required abundant sources and extensivechromatographic separation (Rudd et al., Biochem., 33:17-22 (1994)).Neoglycoproteins (Krantz et al., Biochem., 15:3963-3968 (1976)), formedvia unnatural linkages between sugars and proteins, provide aninvaluable alternative source of carbohydrate-protein conjugates (Forreviews see Stowell et al., Adv. Carbohydr. Chem. Biochem., 37:225-281(1980); Neoglycoconjugates: Preparation and Applications, Lee et al.,Eds., Academic Press, London (1994); Abelson et al., Methods Enzymol.,242: (1994); Lee et al., Methods Enzymol., 247: (1994); Bovin et al.,Chem. Soc. Rev., 24:413-421 (1995)). In particular, chemicalglycosylation allows control of the glycan structure and the nature ofthe sugar-protein bond. However, despite these advantages, existingmethods for their preparation (Stowell et al., Adv. Carbohydr. Chem.Biochem., 37:225-281 (1980)) typically generate mixtures. In addition,these techniques may alter the overall charge of the protein (Lemieux etal., J. Am. Chem. Soc., 97:4076-4083 (1975); Kobayashi et al., MethodsEnzymol., 247:409-418 (1994)) or destroy the cyclic nature of glycansintroduced (Gray, Arch. Biochem. Biophys., 163:426-428 (1974)). Forexample, the reductive amination of lactose with bovine serum albumin(BSA) caused indiscriminate modification of lysine residues through theformation of acyclic amines introduced (Gray, Arch. Biochem. Biophys.,163:426-428 (1974)). Advances in the site-specific glycosylation of BSAhave been made (Davis et al., Tetrahedron Lett., 32:6793-6796 (1991);Wong et al. Biochem. J., 300:843-850 (1994); Macindoe et al., J. Chem.Soc., Chem. Commun. 847-848 (1998)). However, these methods rely uponmodification of an existing cysteine in BSA and, as such, allow noflexibility in the choice of glycosylation site. Glycoproteins occurnaturally as complex mixtures of differently glycosylated forms whichare difficult to separate. To explore their properties, there is a needfor homogenous sources of carbohydrate-protein conjugates. Existingmethods typically generate product protein mixtures of poorlycharacterized composition, with little or no control over the site orlevel of glycosylation.

[0009] The present invention is directed to overcoming thesedeficiencies.

SUMMARY OF THE INVENTION

[0010] It is an object of the present invention to provide for novelglycosylated proteins.

[0011] It is a further object of the invention to provide for novelglycosylated proteins which have modified or improved functionalcharacteristics.

[0012] It is a further object of the invention to provide for a methodof producing glycosylated proteins which have well defined properties,for example, by having predetermined glycosylation patterns.

[0013] According to the present invention, a method is provided whereinthe glycosylation pattern of a protein is modified in a predictable andrepeatable manner. Generally, the modification of the protein occurs viareaction of a cysteine residue in the protein with a glycosylatedthiosulfonate.

[0014] Thus, in one composition aspect of the present invention, achemically modified mutant protein is provided, wherein said mutantprotein differs from a precursor protein by virtue of having a cysteineresidue substituted for a residue other than-cysteine in said precursorprotein, the substituted cysteine residue being subsequently modified byreacting said cysteine residue with a glycosylated thiosulfonate.Preferably, the glycosylated thiosulfonate is an alkylthiosulfonate,most preferably a methanethiosulfonate.

[0015] In a method aspect of the present invention, a method ofproducing a chemically modified mutant protein is provided comprisingthe steps of: (a) providing a precursor protein; (b) substituting anamino acid residue other than cysteine in said precursor protein with acysteine; (c) reacting said substituted cysteine with a glycosylatedthiosulfonate, said glycosylated thiosulfonate comprising a carbohydratemoiety; and (d) obtaining a modified glycosylated protein wherein saidsubstituted cysteine comprises a carbohydrate moiety attached thereto.Preferably, the glycosylated thiosulfonate is an alkylthiosulfonate,most preferably, a methanethiosulfonate. Also preferably, thesubstitution in said precursor protein is obtained by using recombinantDNA techniques by modifying a DNA encoding said precursor protein tocomprise DNA encoding a cysteine at a desired location within theprotein.

[0016] The present invention also relates to novel glycosylatedthiosulfonates. In a preferred embodiment, the glycosylatedthiosulfonate is a methanethiosulfonate. In a most preferred embodiment,the glycosylated methanethiosulfonate comprises a chemical structureincluding:

[0017] where R comprises -β-Glc, -Et-β-Gal, -Et-β-Glc, -Et-α-Glc,-Et-α-Man, -Et-Lac, -β-Glc(Ac)₂, -β-Glc(Ac)₃, -β-Glc(Ac)₄,-Et-α-Glc(Ac)₂, -Et-α-Glc(Ac)₃, -Et-α-Glc(Ac)₄, -Et-β-Glc(Ac)₂,-Et-β-Glc(Ac)₃, -Et-β-Glc(Ac)₄, -Et-α-Man(Ac)₃, -Et-α-Man(Ac)₄,-Et-β-Gal(Ac)₃, -Et-β-Gal(Ac)₄, -Et-Lac(Ac)₅, -Et-Lac(Ac)₆, or-Et-Lac(Ac)₇.

[0018] Another aspect of the present invention is a method of modifyingthe functional characteristics of a protein including reacting theprotein with a glycosylated thiosulfonate reagent under conditionseffective to produce a glycoprotein with altered functionalcharacteristics as compared to the protein. Accordingly, the presentinvention provides for modified protein, wherein the protein comprises awholly or partially predetermined glycosylation pattern which differsfrom the glycosylation pattern of the protein in its precursor, natural,or wild type state and a method for producing such a modified protein.

[0019] The present invention also relates to methods of determining thestructure-function relationships of chemically modified mutant proteins.One method includes providing first and second chemically modifiedmutant proteins of the present invention, wherein the glycosylationpattern of the second chemically modified mutant protein differs fromthe glycosylation pattern of the first chemically modified mutantprotein, evaluating a functional characteristic of the first and secondchemically modified mutant proteins, and correlating the functionalcharacteristic of the first and second chemically modified mutantproteins with the structures of the first and second chemically modifiedmutant proteins. Another method involves providing first and secondchemically modified mutant proteins of the present invention, wherein atleast one different cysteine residue in the second chemically modifiedmutant protein is modified by reacting said cysteine residue with aglycosylated thiosulfonate, evaluating a functional characteristic ofthe first and second chemically modified mutant proteins, andcorrelating the functional characteristic of the first and secondchemically modified mutant proteins with the structures of the first andsecond chemically modified mutant proteins.

[0020] The chemically modified mutant proteins of the present inventionprovide an alternative to site-directed mutagenesis and chemicalmodification for introducing unnatural amino acids into proteins.Moreover, the methods of the present invention allow the preparation ofpure glycoproteins (i.e., not mixtures) with predetermined and uniquestructures. These glycoproteins can then be used to determinestructure-function relationships (e.g., structure-activity relationships(“SARs”)) of non-natural variants of the proteins.

[0021] An advantage of the present invention is that it is possible tointroduce predetermined glycosylation patterns into proteins in a simpleand repeatable manner. This advantage provides an ability to modifycritical protein characteristics such as partitioning, solubility,cell-cell signaling, catalytic activity, biological activity andpharmacological activity. Additionally, the methods of the presentinvention provide for a mechanism of “masking” certain chemically orbiologically important protein sites, for example, sites which arecritical for immunological or allergenic response or sites which arecritical to proteolytic degradation of the modified protein.

[0022] Another advantage of the present invention is the ability toglycosylate a protein which is not generally glycosylated, or to modifythe glycosylation pattern of a protein which is generally glycosylated.

[0023] Another advantage of the present invention is to produce enzymeswhich have altered catalytic activity. In one specific example, theinventors herein have shown that it is possible to modify the substratespecificity of a protease to increase the esterase activity as comparedto the amidase activity. Similarly, modifications of substratespecificity would be expected when utilizing the present invention withother enzymes.

[0024] These and other advantages of the present invention are describedin more detail in the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 shows dendrimer methanethiosulfonate (“MTS”) reagents.

[0026]FIG. 2 shows the synthesis of a first generation glycodendrimerreagent which bears two D-mannose units on its termini and has one armas a MTS which can be attached to a subtilisin Bacillus lentus cysteinemutant.

[0027]FIG. 3 shows the synthesis of highly-functionalizedglycodendrimer-protein conjugates.

[0028]FIG. 4 shows two parallel synthetic approaches to modification ofsubtilisin Bacillus lentus with dendrimers. Both approaches allow theuse of a large library of methanethiosulfonate reagents (R—SSO₂Me) tocap the dendrimeric branches. The routes shown allow for the preparationof both dimeric and trimeric dendrimers.

[0029]FIG. 5 shows peptide coupling catalyzed by an enzyme.

[0030]FIG. 6 shows the preparation of two types of glycosylatingreagents from D-glucose (2a): the anomeric methanethiosulfonate 1alentus and the ethyl-tethered methanethiosulfonates 1b, c, g, h.

[0031]FIG. 7 shows the preparation of the α-D-manno-MTS reagents 1d and1i, which are epimeric at C-2 relative to 1b and 1g, respectively, andthe β-D-galacto-MTS reagents 1e and 1j, epimeric at C-4 relative to 1cand 1h, respectively.

[0032]FIG. 8 shows the reaction of the thiol residue of cysteineintroduced into subtilisin Bacillus lentus withglycomethanethiosulfonate reagents.

[0033]FIG. 9 shows the reaction of2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl methanethiosulfonate (1a) withsubtilisin Bacillus lentus-N62C. -S156C, -S166C, -L217C.

[0034] FIGS. 10A-D show deprotected alycan structure-proteolyticactivity SARs of subtilisin Bacillus lentus cysteine mutants and glycoslated chemically modified mutant enzymes (“CMMs”) relative to wild-type(“WT”). A break in the axis indicates that the value was not determined.At position 62, glycosylation partially restores the decrease ink_(cat)/K_(M) caused by mutation to cysteine (R=H) (FIG. 10A). Atposition 217, the 4-fold decrease in activity caused by mutation (R=H)is amplified to 6-fold lower than WT by glycosylation with untetheredS-β-Glc, but reduced to around 2.5-fold lower than WT by glycosylationwith ethyl-tethered glycans (b-f) (FIG. 10B). At position 156, an arcedvariation in activity reaches a 3-fold lower than WT minimumk_(cat)/K_(M) at bulky lacto-CMM S156C-S-f (FIG. 10C). At position 166,the 2.5-fold decrease in k_(cat)/K_(M) caused by mutation is amplifiedby glycosylation. k_(cat)/K_(M), decreases monotonically from S166C(R=H) to a value that is 3.8-fold lower than WT for S166C-S-f (FIG.10D).

[0035] FIGS. 11A-D show the acetylated glycan structure-proteolyticactivity SARs of glycosylated chemically modified mutant enzymesrelative to WT. For each glycan the number of acetate groups present isindicated by a label on the corresponding bar. A break in the axisindicates that the value was not determined. At positions 62 (FIG. 11A),217 (FIG. 11B), and 166 (FIG. 11D) an alternating trend in activity isobserved as a result of the opposite effects of acetylation uponk_(cat)/K_(M), according to anomeric stereochemistry (see FIG. 12). Thisresults in a k_(cat)/K_(M), for N62C-S-g that is 1.1-fold higher thanWT. At position 156 (FIG. 11C), variations are slight and this isconsistent with its surface exposed orientation.

[0036] FIGS. 12A-D show the variation in proteolytic activity ofglycosylated chemically modified mutant enzymes of subtilisin Bacilluslentus upon acetylation of glycans. Comparison of the activity ofacetylated with fully deprotected chemically modified mutant enzymesshows that at positions 62 (FIG. 12A) and 217 (FIG. 12B) acetylationenhances the activity of (x-tethered chemically modified mutant enzymesbut decreases that of -tethered. In contrast, at position 166 (FIG.12D), acetylation decreases the activity of ax-tethered CMMs butincreases that of P-tethered. Consistent with its surface exposedorientation, changes at position 156 (FIG. 12C) are modest. For eachglycan the number of acetate groups present is indicated by a label onthe corresponding bar. A break in the axis indicates that the value wasnot determined.

[0037] FIGS. 13A-D show esterase k_(cat)/K_(M)s for deprotectedglyco-CMMs relative to WT. At position 62 (FIG. 13A), in the S₁ pocket,glycosylation leads to a series of enzymes that have similar activitiesthat are 1.3- to 1.9-fold greater than WT. At position 217 (FIG. 13B),in the S₁′ pocket, glycosylation also increases k_(cat)/K_(M), to amaximum 3.5-fold greater than WT for L217C-SEtGal (-e). At position 156(FIG. 13C), in the S₁ pocket, glycosylation leads to a reduction ink_(cat)/K_(M). At position 166 (FIG. 13D), in the S₁ pocket, thedramatic loss of activity upon mutation to cysteine (R=H) is restored byglycosylation. All five S166C deprotected glyco-CMMs have similark_(cat)/,K_(M)s that are 1.1- to 1.4-fold lower than WT.

[0038] FIGS. 14A-D show the effect of acetylation on k_(cat)/K_(M) ofglyco-CMMs. For each glycan the number of acetate groups present isindicated by a label on the corresponding bar. A break in the axisindicates that the value was not determined. At positions 62 (FIG. 14A),in the S₂ pocket, and 217 (FIG. 14B), in the S₁′ pocket, the effect ofacetylation is dependent on anomeric stereochemistry. At both sites,acetylation of β-linked sugars (-b,-d) leads to an increase ink_(cat/)K_(M), whereas k_(cat)/K_(M) is decreased for β-linked sugars(-c,-e,-f). k_(cat)/K_(M) also increases as the number of acetatesincreases. Consistent with the surface-exposed nature of its side chain,acetylation at position 156 (FIG. 14C), in the S₁ pocket, has verylittle effect on k_(cat)/K_(M). At position 166 (FIG. 14D), in the S₁pocket, the effects of acetylation are opposite to those observed atpositions 62 and 217. Acetylation increases k_(cat)/K_(M) of W-linkedglyco-CMMs (-c,-e), while causing a decrease for the β-linked gtyco-CMMs(-b,-d).

[0039] FIGS. 15A-D show the E/A of deprotected glyco-CMMs relative toWT.

[0040] A break in the axis indicates that the value was not determined.Glycosylation with deprotected reagents 1b-f increases the E/A ratio inall cases. The greatest effects are observed at positions 62, in the S₂pocket (FIG. 15A), and 217, in the S₁′ pocket (FIG. 15B), where thelargest increases in E/A result in values up to 10.9-fold greater thanWT. At both sites the E/A is higher for the β-linked glyco-CMMs than theα-linked ones. At positions 156 (FIG. 15C) and 166 (FIG. 15D), in the S₁pocket, there is little variation in E/A.

[0041] FIGS. 16A-D show the effect of introducing acetylated glycans onE/A. For each glycan the number of acetate groups present is indicatedby a label on the corresponding bar. A break in the axis indicates thatthe value was not determined. Values greater than zero indicateacetylation increases E/A, negative values denote a reduction in E/Aupon acetylation.

[0042]FIG. 17 shows the modeling of the high esterase activity ofL217C-S-Glc(Ac)₃. A minimized structure of L217C-S-Glc(Ac), of theactive site of SBL showing the catalytic residues Ser221, His64. Thecarbon atoms of the triacetyated D-glucose moiety, which is bound toLeu217 via a disulfide bond, are numbered. The phenyl ring of productAAPF occupies the S₁ binding site and forms a crucial hydrogen bond(1.72 Å) to Wat127. This water molecule is further stabilized by asecond hydrogen bond (1.89 Å) to the carbonyl O of the C-2 acetate groupof glucose.

[0043]FIG. 18 shows the proposed acyl-enzyme intermediate ofL217C-S-Glc(Ac)₃. The carboxy terminus of AAPF forms a bond to the O_(γ)atom of Ser221. Wat127 acting as the crucial deacylating nucleophilicwater molecule, is stabilized through its hydrogen bond to the carbonylgroup of the C-2 acetate group.

DETAILED DESCRIPTION OF THE INVENTION

[0044] According to the present invention, a method is provided whereinthe glycosylation pattern of a protein is modified in a predictable andrepeatable manner. Generally, the modification of the protein occurs viareaction of a cysteine residue in the protein with a glycosylatedthiosulfonate.

[0045] Thus, in one composition aspect of the present invention, achemically modified mutant protein is provided, wherein said mutantprotein differs from a precursor protein by virtue of having a cysteineresidue substituted for a residue other than cysteine in said precursorprotein, the substituted cysteine residue being subsequently modified byreacting said cysteine residue with a glycosylated thiosulfonate.Preferably, the glycosylated thiosulfonate is an alkylthiosulfonate,most preferably, a methanethiosulfonate.

[0046] In a method aspect of the present invention, a method ofproducing a chemically modified mutant protein is provided comprisingthe steps of: (a) providing a precursor protein; (b) substituting anamino acid residue other than cysteine in said precursor protein with acysteine; (c) reacting said substituted cysteine with a glycosylatedthiosulfonate, said glycosylated thiosulfonate comprising a carbohydratemoiety; and (d) obtaining a modified glycosylated protein wherein saidsubstituted cysteine comprises a carbohydrate moiety attached thereto.Preferably, the glycosylated thiosulfonate is an alkylthiosulfonate,most preferably, a methanethiosulfonate. Also preferably, thesubstitution in said precursor protein is obtained by using recombinantDNA techniques by modifying a DNA encoding said precursor protein tocomprise DNA encoding a cysteine at a desired location within theprotein. The amino acid residues to be substituted with cysteineresidues according to the present invention may be replaced usingsite-directed mutagenesis methods or other methods well known in theart. See, for example, PCT Publication No. WO 95/10615, which is herebyincorporated by reference.

[0047] The present invention also relates to a glycosylatedthiosulfonate.

[0048] Preferably, the glycosylated thiosulfonate comprisesmethanethiosulfonate. More preferably, the methanethiosulfonatecomprises the chemical structure:

[0049] where R comprises -β-Glc, -Et-β-Gal, -Et-β-Glc, -Et-α-Glc,-Et-α-Man, -Et-Lac, -β-Glc(Ac)₂, -β-Glc(Ac)₃, -β-Glc(Ac)₄,-Et-α-Glc(Ac)₂, -Et-α-Glc(Ac)₃, -Et-α-Glc(Ac)₄, -Et-β-Glc(Ac)₂,-Et-β-Glc(Ac)₃, -Et-β-Glc(Ac)₄, -Et-α-Man(Ac)₃, -Et-α-Man(Ac)₄,-Et-β-Gal(Ac)₃, -Et-β-Gal(Ac)₄, -Et-Lac(Ac)₅, -Et-Lac(Ac)₆, or-Et-Lac(Ac)₇.

[0050] Another aspect of the present invention is a method of modifyingthe functional characteristics of a protein including providing aprotein and reacting the protein with a glycosylated thiosulfonatereagent under conditions effective to produce a glycoprotein withaltered functional characteristics as compared to the protein.Accordingly, the present invention provides for modified protein,wherein the protein comprises a wholly or partially predeterminedglycosylation pattern which differs from the glycosylation pattern ofthe protein in its precursor, natural. or wild type state and a methodfor producing such a modified protein. As used herein, glycosylationpattern means the composition of a carbohydrate moiety. The presentinvention also relates to methods of determining the structure-functionrelationships of chemically modified mutant proteins. The first methodincludes providing first and second chemically modified mutant proteinsof the present invention, wherein the glycosylation pattern of thesecond chemically modified mutant protein differs from the glycosylationpattern of the first chemically modified mutant protein, evaluating afunctional characteristic of the first and second chemically modifiedmutant proteins. and correlating the functional characteristic of thefirst and second chemically modified mutant proteins with the structuresof the first and second chemically modified mutant proteins. The secondmethod involves providing first and second chemically modified mutantproteins of the present invention, wherein at least one differentcysteine residue in the second chemically modified mutant protein ismodified by reacting said cysteine residue with a glycosylatedthiosulfonate, evaluating a functional characteristic of the first andsecond chemically modified mutant proteins, and correlating thefunctional characteristic of the first and second chemically modifiedmutant proteins with the structures of the first and second chemicallymodified mutant proteins.

[0051] The chemically modified mutant proteins of the present inventionprovide a valuable source of carbohydrate-protein conjugates. Moreover,the methods of the present invention allow the preparation of pure andglycoproteins (i.e., not mixtures) with predetermined and uniquestructures. These glycoproteins can then be used to determinestructure-function relationships (e.g., structure-activity relationships(“SARs”)) of non-natural variants of the proteins.

[0052] The protein of the invention may be any protein for which amodification of the glycosylation pattern thereof may be desirable. Forexample, proteins which are naturally not glycosylated may beglycosylated via the invention. Similarly, proteins which exist in anaturally glycosylated form may be modified so that the glycosylationpattern confers improved or desirable properties to the protein.Specifically, proteins useful in the present invention are those inwhich alycosylation plays a role in functional characteristics such as,for example, biological activity, chemical activity, pharmacologicalactivity or immunological activity.

[0053] Glycosylated proteins as referred to herein means moieties havingcarbohydrate components which are present on proteins, peptides, oramino acids. In the present invention, the glucosylation is provided,for example, as a result of reaction of the glycosylated thiosulfonatewith the thiol hydrogen of a cysteine residue thereby producing an aminoacid residue which has bound thereto the carbohydrate component presenton the glycosylated thiosulfonate.

[0054] Another aspect of the present invention is a method of modifyingthe functional characteristics of a protein including providing aprotein and reacting the protein with a glycosylated thiosulfonatereagent under conditions effective to produce a glycoprotein withaltered functional characteristics as compared to the protein.

[0055] The functional characteristics of a protein which may be alteredby the present invention include. but are not limited to, enzymaticactivity, the effect on a human or animal body, the ability to act as avaccine, the tertiary structure (i.e., how the protein folds), whetherit is allergenic, its solubility, its signaling effects, its biologicalactivity, and its pharmacological activity (Paulson, “Glycoproteins:What are the Sugar Chains For?”, Trends in Biochem. Sciences, 14,272-276(1989), which is hereby incorporated by reference). The use ofglycosylated thiosulfonates as thiol-specific modifying reagents in themethod of the present invention allows virtually unlimited alterationsof protein residues. In addition, this method allows the production ofpure glycoproteins with predetermined and unique structures and,therefore, unique functional characteristics, with control over both thesite and level of glycosylation. In particular, the method of modifyingthe functional characteristics of a protein allows the preparation ofsingle glycoforms through regio- and glycan-specific proteinglycosylation at predetermined sites. Such advantages provide an arrayof options with respect to modification of protein properties which didnot exist in the prior art. The ability to produce proteins having veryspecific and predictable glycosylation patterns will enable theproduction of proteins which have known and quantifiable effects inchemical, pharmaceutical, immunological, or catalytic performance- Forexample, with knowledge of a specific problematic epitope, it would bepossible to construct a modified protein according to the presentinvention in which the epitope is masked by a carbohydrate moiety, thusreducing its allergenic or immunogenic response in a subject. As anotherexample, where the solubility of a protein is problematic in terms ofrecovery or formulation in a pharmaceutical or industrial application,it may be possible, utilizing the present invention. to produce aprotein which has altered solubility profiles thus producing a moredesirable protein product. As another example, if a protein hasparticular problem of being proteolytically unstable in the environmentin which it is to be used, then it may be possible to mask theproteolytic cleavage sites in the protein using the present invention tocover up such a site with a carbohydrate moiety. These examples aremerely a few of the many applications of the present invention toproduce improved proteins.

[0056] In a preferred embodiment, the protein is an enzyme. The term“enzyme” includes proteins that are capable of catalyzing chemicalchanges in other substances without being changed themselves. Theenzymes can be wild-type enzymes or variant enzymes. Enzymes within thescope of the present invention include pullulanases, proteases,cellulases, amylases, isomerases, lipases, oxidases, and reductases.Preferably, the enzyme is a protease. The enzyme can be a wild-type ormutant protease. Wild-type proteases can be isolated from, for example,Bacillus lentus or Bacillus amyloliquefctciens (also referred to asBPN′). Mutant proteases can be made according to the teachings of, forexample, PCT Publication Nos. WO 95/10615 and WO 91/06637, which arehereby incorporated by reference. Functional characteristics of enzymeswhich are suitable for modification according to the present inventioninclude, for example, enzymatic activity, solubility, partitioning,cell-cell signaling, substrate specificity, substrate binding, stabilityto temperature and reagents, ability to mask an antigenic site,physiological functions, and pharmaceutical functions (Paulson,“Glycoproteins: What are the Sugar Chains For?”, Trends in Biochem.Sciences, 14:272-276 (1989), which is hereby incorporated by reference).

[0057] The protein is modified so that a non-cysteine residue issubstituted with a cysteine residue, preferably by recombinant means.Preferably, the amino acids replaced in the protein by cysteines areselected from the group consisting of asparagine, leucine, or serine.

[0058] The terms “'thiol side chain group,” “thiol containing group,”and “thiol side chain” are terms which are can be used interchangeablyand include groups that are used to replace the thiol hydrogen of acysteine used to replace one of the amino acids in a protein. Commonly,the thiol side chain group includes a sulfur through which the thiolside chain groups defined above are attached to the thiol sulfur of thecysteine.

[0059] The glycosylated thiosulfonates of the invention are those whichare capable of reacting with a thiol hydrogen of a cysteine to produce aglycosylated amino acid residue. By glycosylated is meant that thethiosulfonate has bound thereto a sugar or carbohydrate moiety which canbe transferred to a protein pursuant to the present invention.Preferably, the glycosylated thiosulfonates are glycosylatedalkylthiosulfonates, most preferably, glycosylatedmethanethiosulfonates. Such glycosylated methanethiosulfonate have thegeneral formula:

[0060] In particularly preferred embodiment, the methanethiosulfonatecomprises an R group which comprises: -β-Glc, -Et-β-Gal, -Et-β-Glc,-Et-α-Glc, -Et-α-Man, -Et-Lac, -β-Glc(Ac)₂, -β-Glc(Ac)₃, -β-Glc(Ac)₄,-Et-α-Glc(Ac)₂, -Et-α-Glc(Ac)₃, -Et-α-Glc(Ac)₄, -Et-β-Glc(Ac)₂,-Et-β-Glc(Ac)₃, -Et-β-Glc(Ac)₄, -Et-α-Man(Ac)₃, -Et-α-Man(Ac)₄,-Et-β-Gal(Ac)₃, -Et-β-Gal(Ac)₄, -Et-Lac(Ac)₅, -Et-Lac(Ac)₆, or-Et-Lac(Ac)₇.

[0061] In a preferred embodiment, the carbohydrate moiety of the presentinvention is a dendrimer moiety. Multiple functionalization ofchemically modified mutant proteins can be achieved by dendrimerapproaches, whereby multiple-branched linking structures can be employedto create poly-functionalized chemically modified mutant proteins.

[0062] Highly branched molecules or dendrimers were first synthesized byVögtle in 1978 (Buhleier et al., Synthesis, 155-158 (1978), which ishereby incorporated by reference). The attachment of identical buildingblocks that contain branching sites to a central core may be achievedwith a high degree of homogeneity and control. Each branch contains afunctional group which, after chemical alteration, may be connected toyet another branching building block. In this manner, layer after layerof branching rapidly generates highly-functionalized molecules.

[0063] For instance, multiple glycosylation, including multiplemannose-containing chemically modified mutant proteins, and varied sugarmoieties, can be created. The dendrimer reagent structures would includemethanethiosulfonates with simple branching such as:

[0064] derived from pentaerythritol, to very complex branched dendrimerreagents (see FIG. 1).

[0065] In particular, a first generation glycodendrimer reagent issynthesized as shown in FIG. 2. This approach can be extended to coverlarger dendrimers. More specifically, by leaving one “arm” of theglycodendrimer free for conversion to a methanethiosulfonate, theremaining arms can be further branched to synthesizehighly-functionalized glycodendrimer reagents as shown in FIG. 3.Through further branching and by using different carbohydrates, thisconcept can be extended to virtually unlimited levels.

[0066] A flexible synthetic strategy for the synthesis of coredendrimeric methanethiosulfonate building blocks that may be used eitherin situ or before modification to construct dendrimers is shown in FIG.4.

[0067] The present invention also relates to glycosylated thiosulfonatecompositions. Preferably the glycosylated thiosulfonates aremethanethiosulfonates and comprise a chemical structure:

[0068] wherein R comprises -β-Glc, -Et-β-Gal, -Et-β-Glc, -Et-α-Glc,-Et-α-Man, -Et-Lac, -β-Glc(Ac)₂, -β-Glc(Ac)₃, -β-Glc(Ac)₄,-Et-α-Glc(Ac)₂, -Et-α-Glc(Ac)₃, -Et-α-Glc(Ac)₄, -Et-β-Glc(Ac)₂,-Et-β-Glc(Ac)₃, -Et-β-Glc(Ac)₄, -Et-α-Man(Ac)₃, -Et-α-Man(Ac)₄,-Et-β-Gal(Ac)₃, -Et-β-Gal(Ac)₄, -Et-Lac(Ac)₅, -Et-Lac(Ac)₆, or-Et-Lac(Ac)₇.

[0069] The present invention also relates to a method of determining thestructure-function relationships of chemically modified mutant proteins.This method involves providing first and second chemically modifiedmutant proteins of the present invention, wherein the glycosylationpattern of the second chemically modified mutant protein is differentfrom the glycosylation pattern of the first chemically modified mutantprotein, evaluating a functional characteristic of the first and secondchemically modified mutant proteins, and correlating the functionalcharacteristic of the first and second chemically modified mutantproteins with the structures of the first and second chemically modifiedmutant proteins.

[0070] Evaluating a functional characteristic of the first and secondchemically modified mutant protein includes testing for functionalcharacteristics including, but not limited to, stability to temperatureand reagents, solubility, partitioning, enzymatic activity, cell-cellsignaling, substrate specificity, substrate binding, ability to mask anantigenic site, physiological functions, and pharmaceutical functions(Paulson, “Glycoproteins: What are the Sugar Chains For?”, Trends inBiochem. Sciences, 14:272-276 (1989), which is hereby incorporated byreference).

[0071] Another aspect of the present invention is a second method ofdetermining the structure-function relationships of chemically modifiedmutant proteins. This method involves providing first and secondchemically modified mutant proteins of the present invention, wherein atleast one different cysteine residue in the second chemically modifiedmutant protein is modified by reacting said cysteine residue with aglycosylated thiosulfonate, evaluating a functional characteristic ofthe first and second chemically modified mutant proteins, andcorrelating the functional characteristic of the first and secondchemically modified mutant proteins with the structures of the first andsecond chemically modified mutant proteins.

[0072] By way of example to illustrate some of its advantages, thefollowing discussion will focus on certain proteases which are modifiedaccording to the methods of the present invention. Alkaline serineproteases (subtilisins) are finding increasing use in biocatalysis,particularly in chiral resolution, regioselective acylation ofpolyfunctional compounds, peptide coupling, and glycopeptide synthesis.As shown in FIG. 5, subtilisins can catalyze peptide bond formationstarting from an ester substrate, by first forming an acyl enzymeintermediate which then reacts with a primary amine to form the peptideproduct. This application requires high esterase activity to promoteacyl enzyme formation and low amidase activity to minimize hydrolysis ofthe peptide bond of the desired product. Generally, subtilisins do notmeet these requirements. However, the improvement of the esterase toamidase selectivities of subtilisins has been a long sought after goal.By using the methods provided for in the present invention, it ispossible to produce subtilisins which have advantageous properties.

[0073] The inventors in the present case used site specific mutagenesisto modify certain residues and introduce additional cysteine residueswithin subtilisin which would then serve to react with a glycosylatedmethanethiosulfonate to produce a glycosylation point at the introducedcysteine. Bacillus lentus subtilisin was selected for illustrativepurposes because it does not contain a natural cysteine and is notnaturally glycosylated.

[0074] The substrate binding site of an enzyme consists of a series ofsubsites across the surface of the enzyme. The portion of substrate thatcorresponds to the subsites are labeled P and the subsites are labeledS. By convention, the subsites are labeled S₁, S₂, S₃, S₄, S₁′, and S₂′.A discussion of subsites can be found in Berger et al., Phil. Trans.Roy. Soc. Lond. B, 257:249-264 (1970), Siezen et al., ProteinEngineering, 4:719-737 (1991), and Fersht, Enzyme Structure andMechanism, 2 ed., Freeman: New York, 29-30 (1985), which are herebyincorporated by reference.

[0075] In the present illustration, the S₁, S₁′, or S₂ subsites wereselected as suitable targets for modification. In particular, the aminoacids corresponding to N62, L217, S156, and S166 in naturally-occurringsubtilisin from Bacillus amyloliquefaciens or to equivalent amino acidresidues in other subtilisins, such as Bacillus lentus subtilisin, wereselected for modification to cysteine. The mutated subtilisin wasproduced through standard site directed mutagenesis techniques -and theobtained mutant subtilisin was reacted with certain glycosylatedalkylthiosulfonates, particularly glycosylated methanethiosulfonates, asprovided in the examples appended hereto. —Enzymatic peptide coupling isan attractive method for preparation of a variety of peptides, becausethis method requires minimal protection of the substrate. proceeds undermild conditions, and does not cause racemization. Wong et al., Enzymesin Synthetic Organic Chemistry, Pergamon Press: Oxford, 41-130 (1994),which is hereby incorporated by reference. In spite of these advantages,two major problems have limited the use of serine proteases in peptidesynthesis. One is their efficient proteolytic (amidase) activity whichcauses hydrolysis of the coupling product, and the other is theirstringent structural specificity and stereospecificity.

[0076] Surprisingly, it was found that the chemically modified mutantsubtilisins of the present invention have altered esterase-to-amidaseactivity as compared to the precursor enzyme. Increasing theesterase-to-amidase ratio enables the use of the enzyme to moreefficiently catalyze peptide synthesis. In particular, subtilisins cancatalyze peptide bond formation starting from an ester substrate (i.e.an acyl donor), by first forming an acyl enzyme intermediate which thenreacts with a primary amine (i.e. an acyl acceptor) to form the peptideproduct, as shown in FIG. 5. This reaction thus requires high esteraseactivity to promote acyl enzyme formation and, then, low amidaseactivity to minimize hydrolysis of the peptide bond of the desiredproduct. The chemically modified mutant subtilisins produced accordingto the present invention show an increased esterase-to-amidase ratio,without reducing the absolute esterase activity of the enzyme. Inaddition, certain modified enzymes of the present invention even show aconcomitant increase in the absolute esterase activity.

[0077] Therefore, an unexpected- benefit of subtilisins which aremodified according to the present invention is that they can be used inorganic synthesis to, for example, catalyze a desired reaction and/orfavor a certain stereoselectivity. See e.g., Noritomi et al. Biotech.Bioeng. 51:95-99 (1996); Dabulis et al. Biotech. Bioeng. 41:566-571(1993), and Fitzpatrick et al. J. Am. Chem. Soc. 113:3166-3171 (1991),which are hereby incorporated by reference.

[0078] Proteins obtained using the methods provided herein may be usedin any application in which it is desired to use such proteins, wherehaving modified functional capabilities is advantageous. Thus, proteinsmodified as provided herein may be used in the medical field forpharmaceutical compositions and in diagnostic preparations.Additionally, proteins such as enzymes which are modified according tothe present invention may be used in applications which are generallyknown for such enzymes including industrial applications such ascleaning products, textile processing, feed modification, foodmodification, brewing of grain beverages. starch processing, asantimicrobials, and in personal care formulations. Moreover, the uniquefunctionalities made possible by the present invention may result inuses for proteins which have not heretofore been recognized as feasible.

EXAMPLES Example 1 Preparation of Methanethiosulfonate (“NMTS”) Reagents

[0079] The preparation of NaSSO₂CH₃ (Kenyon et al., Methods Enzymol.,47:407-430 (1977), which is hereby incorporated by reference) has beendescribed previously (Berglund et al., J. Am. Chem. Soc., 119:5265-5266(1997), which is hereby incorporated by reference). Acetobromoglucose(3) (See FIG. 6) (prepared from D-glucose according to Scheurer et al.,J. Am. Chem. Soc., 76:3224 (1954), which is hereby incorporated byreference) in 73% yield, pentaacetylglucose (prepared from thecorresponding parent carbohydrates according to the method of Verley etal., Ber. Dtsch. Chem. Ges., 34:3354-3358 (1901), which is herebyincorporated by reference, and purified by flash chromatography) in 99%yield, 5d (See FIG. 7) (prepared from the corresponding parentcarbohydrates according to the method of Verley et al., Ber. Dtsch.Chem. Ges., 34:3354-3358 (1901), which is hereby incorporated byreference, and purified by flash chromatography) in 92% yield, 5e (SeeFIG. 7) (prepared from the corresponding parent carbohydrates accordingto the method of Verley et al., Ber. Dtsch. Chem. Ges., 34:3354-3358(1901), which is hereby incorporated by reference, and purified by flashchromatography) in 99% yield, 5f (See FIG. 7) (prepared from lactoseaccording to the method of Hudson et al., J. Am. Chem. Soc.,37:1270-1275 (1915), which is hereby incorporated by reference, andpurified by flash chromatography in 82% yield) were prepared accordingto literature methods. N,N-dimethylformamide (“DMF”) was distilled underN₂ from CaH₂ and stored over molecular sieve under N₂ before use.Methanol was distilled from Mg/I₂ under N₂ immediately prior to use.Br(CH₂)₂OH was stood over and distilled from CaO under reduced pressureand stored under N₂ prior to use. All other chemicals were used asreceived from Sigma-Aldrich (St. Louis, Mo.) or Baker (Phillipsburg,N.J.). All flash chromatography was performed using silica gel (Whatman,60 Å, 230-400 Mesh, Clifton, N.J.). Melting points were determined usingan Electrothermal IA9000 series digital melting point apparatus and areuncorrected. IR spectra were recorded on Bomem MB or Perkin-Elmer FTIRSpectrum 1000 spectrophotometers. ¹H NMR and ¹³C NMR spectra wererecorded on Varian Gemini 200, Unity 400 or Unity 500 NMR spectrometersat the frequencies indicated. Where indicated, NMR peak assignments weremade using Correlation Spectroscopy (“COSY”) or Distortionless Enhancedby Polarization Transfer (“DEPT”) experiments, all others aresubjective. All chemical shifts were referenced to residual solvent asan internal standard; for ¹³C NMR in D₂O 1,4-dioxan (67.6 ppm) was used.ES-MS data were acquired using a PE SCIEX API III Biomolecular massspectrometer. All HRMS data were acquired using Micromass 70-250S orMicromass ZAB-SE mass spectrometers according to the ionization methodsindicated. Solvents were removed in vacuo.

[0080] Preparation of 2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosylmethanethiosulfonate (1a).

[0081] Initial approaches to untethered glyco-MTS reagents similar intype to 1a (See FIG. 8) were based upon Danishefsky's glycal methodology(Halcomb et al., J. Am. Chem. Soc., 111:6661-6666 (1989), which ishereby incorporated by reference). Tris-TBS glucal was preparedaccording to the method of Lesimple et al., Tetrahedron Lett.,27:6201-6204 (1986), which is hereby incorporated by reference, andoxidized to tris-TBS protected 1,2-anhydroglucose usingdimethyldioxirane. However, under a variety of conditions and incontrast to the behavior of other sulfur nucleophiles (Gordon et al.,Carbohydr. Res., 206:361-366 (1990); Berkowitz et al, J. Am. Chem. Soc.,114:4518-4529 (1992), which are hereby incorporated by reference),methanethiosulfonate ion failed to open the epoxide moiety of tris-TBSprotected 1,2-anhydro-D-glucose. Deprotection of 1a (See FIG. 8) wasattempted under a variety of conditions, but in all cases led only todecomposition or hydrolysis of the thioglucosidic bond (Zemplen et al.,Ber. Dtsch. Chem. Ges., 56:1705-1710 (1923); Plattner et al., J. Am.Chem. Soc., 94:8613-8615 (1972); Mori et al., Tetrahedron Lett.,20:1329-1332 (1979); Herzig et al., Carbohydr. Res., 153:162-167 (1986);Herzig et al., J. Org. Chem., 51:727-730 (1986); Vekemans et al.,Tetrahedron Lett., 28:2299-2300 (1987); Cinget et al., Synlett, 168-170(1993), which are hereby incorporated by reference).

[0082] Acetobromoglucose (3) (See FIG. 6) (1 g, 2.43 mmol) was added toa solution of NaSSO₂CH₃ (380 mg, 2.84 mmol) in ethanol (4 mL) at 90° C.under N₂. After 20 minutes the resulting suspension was cooled and thesolvent removed. The residue was purified by flash chromatography(EtOAc:hexane, 9:11) and the resulting solid recrystallized from etherto give 1a (See FIG. 8) (674 mg, 63%) as a white solid: mp 151-152° C.melts then decomp. (ether); [α]²⁷ _(D)=−19.0 (c 1.24, CHCl₃); IR (KBr)1749 cm⁻¹ (C═O), 1333, 1140 cm⁻¹ (S—SO₂); ¹H NMR (400 MHz, CDCl₃) δ2.00, 2.04, 2.06, 2.07 (s×4, 3H×4, Ac×4), 3.44 (s, 3H, CH₃SO₂—), 3.82(ddd, J_(4,5) 10.1 Hz, J_(5,6) 5.9 Hz, J_(5,6′) 2.2 Hz, 1H, H-5), 4.08(dd, J_(5,6) 5.9 Hz, J_(6,6′) 12.5 Hz, 1H, H-6), 4.31 (dd, J_(5,6′) 2.2Hz, J_(6,6′) 12.5 Hz, 1H, H-6′), 5.05 (t, J 9.8 Hz, 1H, H-4), 5.07 (dd,J_(1,2) 10.5 Hz, J_(2,3) 9.4 Hz, 1H, H-2), 5.25 (d ,J_(1,2) 10.5 Hz, 1H,H-1), 5.29(t, J 9.3 Hz, 1H, H-3); ¹³C NMR (50 MHz, CDCl₃) δ 20.5, 20.7(CH₃COO—×4), 52.8 (CH₃SO₂—), 61.8, 68.0, 68.7, 73.3, 76.6 (C-2, C-3,C-4, C-5, C-6), 86.4 (C-1), 169.3, 169.3, 169.7, 170.1 (CH₃ COO—×4);HRMS m/z (EI+): Found 443.0636 (M+H⁺); C₁₅H₂₃O₁₁S₂ requires 443.0682.

[0083] Preparation of 2-(2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl)ethylMethanethiosulfonate (1g).

[0084] BF₃.Et₂O (145 μL, 1.1 mmol) was added dropwise to a suspension ofD-glucose (2a) (See FIG. 6) (1.45 g, 8.1 mmol) in Br(CH₂)₂OH (19 mL)under N₂ and the resulting mixture heated to 105° C. After 8 hours, theresulting solution was cooled and the solvent removed. The residue wasdissolved in Ac₂O/pyridine (2:3 v/v, 16 mL) under N₂. After a further 24hours, the reaction solvent was removed and the residue purified byrepeated flash chromatography (EtOAc then EtOAc:hexane, 3:7) to give2-bromoethyl 2,3,4,6-tetra-O-acetyl-α-D-glucopyranoside (4g) (See FIG.6) (1.76 g, 48%) as a colorless oil that crystallized on standing togive a white solid; mp 86-88° C.; [α]²⁵ _(D)=+130.6 (c 0.21, CHCl₃); IR(film) 1749 cm⁻¹ (C═O); ¹H NMR (400 MHz, CDCl₃) δ 2.01, 2.03, 2.07, 2.09(s×4, 3H×4, Ac×4), 3.51 (t, J 5.9 Hz, 2H, —CH₂Br), 3.83 (dt, J_(d) 11.6Hz, J_(t) 5.8 Hz, 1H, —OCHH′—), 3.96 (dt, J_(d) 11.6 Hz, J_(t) 5.8 Hz,1H, —OCHH′—), 4.10 (dd, J_(5,6) 2.2 Hz, J_(6,6′) 12.0 Hz, 1H, H-6), 4.14(ddd, J_(4,5) 10.2 Hz, J_(5,6) 2.2 Hz, J_(5,6′) 4.4 Hz, 1H, H-5), 4.24(dd, J_(5,6′) 4.4 Hz, J_(6,6′ 12.0) Hz, 1H, H-6′), 4.84 (dd, J_(1,2) 3.8Hz, J_(2,3) 10.3 Hz, 1H, H-2), 5.05 (t, J 9.7 Hz, 1H, H-4), 5.14 (d,J_(1,2) 3.8 Hz, 1H, H-1), 5.49 (dd, J_(2,3) 10.3 Hz, J_(3,4) 9.5 Hz, 1H,H-3); ¹³C NMR (100 MHz, CDCl₃) δ 20.6, 20.7 (CH₃COO—×4), 29.9 (—CH₂Br),61.9, 67.8, 68.5, 68.8, 70.0, 70.8 (—OCH₂—, C-2, C-3, C-4, C-5, C-6),96.0 (C-1), 169.6, 170.0, 170.2, 170.6 (CH₃COO—×4); HRMS m/z (FAB+):Found 477.0381 (M+Na⁺); C₁₆H₂₃O₁₀BrNa requires 477.0372. NaSSO₂CH₃ (75mg, 0.56 mmol) was added to a solution of 4g (See FIG. 6) (190 mg, 0.42mmol) in DMF (6 mL) under N₂ and warmed to 50° C. After 21 hours, thesolution was cooled and the solvent removed. The residue was purified byflash chromatography (EtOAc:hexane, 1:1) to give 1g (See FIG. 6) (183mg, 90%) as a colorless oil; [α]²⁷D=+92.1 (c 0.39, CHCl₃); IR (film)1748 cm⁻¹ (C═O), 1322, 1134 cm⁻¹ (S—SO₂); ¹H NMR (400 MHz, CDCl₃) δ2.01, 2.03, 2.07, 2.09 (s×4, 3H×4, Ac×4), 3.41 (t, J 5.7 Hz, 2H,—CH₂S—), 3.41 (s, 3H, CH₃SO₂—), 3.75 (dt, J_(d) 10.8 Hz, J_(t) 5.7 Hz,1H, —OCHH′—), 3.99-4.06 (m, 2H, H-5, —OCHH′′), 4.09 (dd, J_(5,6) 2.4 Hz,J_(6,6′) 12.6 Hz, 1H, H-6), 4.25 (dd, J_(5,6′) 4.6 Hz, J_(6,6′) 12.6 Hz,1H, H-6′), 4.87 (dd, J_(1,2) 3.9 Hz, J_(2,3) 10.3 Hz, 1H, H-2), 5.06 (t,J 9.8 Hz, 1H, H-4), 5.12 (d, J₁₂ 3.9 Hz, 1H, H-1), 5.43 (t, J 9.8 Hz,1H, H-3); ¹³C NMR (100 MHz, CDCl₃) δ 20.6, 20.6, 20.7, 20.7 (CH₃COO—×4),36.0, (—CH₂S—), 50.8 (CH₃SO₂—), 61.8 (—OCH₂—), 67.0, 67.8, 68.3, 69.8,70.7 (C-2, C-3, C-4, C-5, C-6), 96.0 (C-1), 169.5, 170.0, 170.6 (CH₃COO—×4); HRMS m/z (FAB+): Found 487.0946 (M+H⁺); C₁₇H₂₇O₁₂S₂ requires487.0944.

[0085] Preparation of 2-(α-D-Glucopyranosyl)ethyl Methanethiosulfonate(1b).

[0086] A solution of NaOMe (0.1M, 0.3 mL) was added to a suspension of4g (See FIG. 6) (300 mg, 0.66 mmol) in MeOH (3 mL) under N₂ and stirredvigorously. After 6 hours, the resulting solution was passed through aDowex 50W(H⁺) plug (2×1 cm, eluant MeOH) and the solvent removed to give2-bromoethyl α-D-glucopyranoside bromide (4b) (See FIG. 6) (178 mg, 94%)(The synthesis of 4b as an intermediate has been described previously.However, this method gave only a poor yield of product. Nagai et al.,Carbohydr. Res., 190:165-180 (1989), which is hereby incorporated byreference) as a white solid. NaSSO₂CH₃ (100 mg, 0.75 mmol) was added toa solution of 4b (See FIG. 6) (178 mg, 0.62 mmol) in DMF (7 mL) under N₂and warmed to 50° C.

[0087] After 25 hours, the solution was cooled and the solvent removed.The residue was purified by flash chromatography (MeOH:EtOAc, 1:9) togive 1b (See FIG. 6) (144 mg, 73%) as a hygroscopic foam; [α]²⁷_(D)=+109.9 (c 1.11, H₂O); IR (film) 3423 cm⁻¹ (OH), 1309, 1128 cm⁻¹(S—SO₂); ¹H NMR (500 MHz, D₂O, COSY) δ 3.16 (t, J 9.5 Hz, 1H, H-4), 3.28(t, J 5.9 Hz, H, —CH₂S—), 3.30 (s, 3H, CH₃SO₂—), 3.31 (dd, J_(1,2) 3.8Hz, J_(2,3) 9.9 Hz, 1H, H-2), 3.44 (t, J 9.5 Hz, 1H, H-3), 3.47-3.53 (m,2H, H-6, H-6′), 3.58-3.61 (m, 1H, H-5), 3.62 (dt, J_(t) 5.4 Hz, J_(d)10.8 Hz, 1H, —OCHH′—), 3.79 (dt, J_(t) 6.3 Hz, J_(d) 10.8 Hz, 1H,—OCHH′) 4.72 (d, J_(1,2) 3.8 Hz, 1H, H-1); ¹³C NMR (100 MHz, D₂O) δ 36.7(—CH₂S—), 50.7 (CH₃SO₂—), 61.5 (—OCH₂—), 67.2, 70.5, 72.3, 73.2, 74.0(C-2, C-3, C-4, C-5, C-6), 99.4 (C-1); HRMS m/z (FAB+): Found 319.0517(M+H⁺); C₉H₁₉O₈S₂ requires 319.0521.

[0088] Preparation of 2-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl)ethylMethanethiosulfonate (1h).

[0089] BF₃.Et₂O (3.3 mL, 26.0 mmol) was added dropwise over the courseof 15 minutes to a solution of 1,2,3,4,6-penta-O-acetyl-α,β-D-glucose (2g, 5.1 mmol) and Br(CH₂)₂OH (0.45 mL, 6.3 mmol) in CH₂Cl₂ (9 mL) at 0°C. under N₂. After 1.5 hours, the solution was warmed to roomtemperature. After 20 hours the reaction solution was added to ice water(15 mL) and extracted with CH₂Cl₂ (15 mL×3). These extracts werecombined, washed with water (15 mL), sat. NaHCO₃ (aq., 15 mL), water (15mL), dried (MgSO₄), filtered, and the solvent removed. The residue waspurified by flash chromatography (EtOAc:hexane, 1:3) to give2-bromoethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (4h) (See FIG.6) (1.42 g, 61%) as a white solid; mp 118-120° C. (EtOAc/iso-octane)[lit., (Coles et al., J. Am. Chem. Soc., 60:1020-1022 (1938), which ishereby incorporated by reference) mp 117.3° C. (EtOH)]; [α]²⁷ _(D)=−11.9(c 1.65, CHCl₃) [lit., (Helferich et al., Just. Lieb. Ann. Chem.,541:1-16 (1939), which is hereby incorporated by reference) [α]²⁰_(D)=−12.3 (c 0.2, CHCl₃)]; ¹H NMR (200 MHz, CDCl₃) δ 2.00, 2.02, 2.07,2.09 (s×4, 3H×4, Ac×4), 3.42-3.51 (m, 2H), 3.67-3.87 (m, 2H), 4.10-4.31(m, 3H), 4.57 (d, J_(1,2) 8 Hz, 1H, H-1), 4.97-5.27 (m, 3H). NaSSO₂CH₃(260 mg, 1.94 mmol) was added to a solution of 4h (See FIG. 6) (640 mg,1.41 mmol) in DMF (18 mL) under N₂ and warmed to 50° C. After 25 hours,the solution was cooled and the solvent removed. The residue waspurified by flash chromatography (EtOAc:hexane, 1:1) and the resultingsolid recrystallized from EtOAc/hexane to give 1h (See FIG. 6) (544 mg,80%) as a white solid; mp 115-116° C. (EtOAc/hexane); [α]²⁷ _(D)=+5.4 (c1.06. CHCl₃); IR (KBr) 1758, 1741 cm⁻¹ (C═O), 1314, 1133 cm⁻¹ (S—SO₂);¹H NMR (500 MHz, CDCl₃, COSY) δ 1.99, 2.02, 2.06, 2.08 (s×4, 3H×4,Ac×4), 3.30-3.38 (m, 2H, —CH₂S—), 3.34 (s, 3H, CH₃SO₂—), 3.70 (ddd,J_(4,5) 9.9 Hz, J_(5,6) 2.2 Hz, J_(5,6) 4.6 Hz, 1H, H-5), 3.83 (ddd, J5.6 Hz, J 7.4 Hz, J 10.5 Hz, 1H, —OCHH′—), 4.13-4.18 (m, 2H, H-6,—OCHH′—), 4.24 (dd, J_(5,6′) 4.6 Hz, J_(6,6) 12.4 Hz, 1H, H-6′), 4.55(d. J_(1,2) 8.1 Hz, 1H, H-1), 4.98 (dd J_(1,2) 8.1 Hz, J_(2,3) 9.7 Hz,1H, H-2), 5.07 (t, J 9.9 Hz, 1H, H-4), 5.19 (t, J 9.6 Hz, 1H, H-3); ¹³CNMR (125 MHz, CDCl₃ DEPT) δ 20.5, 20.7 (q×2, CH₃COO—×4), 36.0, (t,—CH₂S—), 50.6 (q, CH₃SO₂—), 61.6 (t, —OCH₂—), 68.1, 70.8, 71.9, 72.5(d×4, C-2, C-3, C-4, C-5), 68.4 (t, C-6), 100.8 (d, C-1), 169.3, 170.0,170.5 (s×3, CH₃ COO—×4); HRMS m/z (FAB+): Found 487.0940 (M+H⁺);C₁₇H₂₇O₁₂S₂ requires 487.0944.

[0090] Preparation of 2-(β-D-Glucopyranosyl)ethyl Methanethiosulfonate(1c).

[0091] A solution of NaOMe (0.1M, 0.3 mL) was added to a suspension of4h (See FIG. 6) (300 mg, 0.66 mmol) in MeOH (3 mL) under N₂ and stirredvigorously. After 4 hours, the resulting solution was passed through aDowex 50W(H-) plug (2×1 cm, eluant MeOH) and the solvent removed to give2-bromoethyl β-D-glucopyranoside 4c (See FIG. 6) (176 mg, 93%) as awhite solid that was used directly in the next step. A sample wasrecrystallized from EtOH/EtOAc to give a colorless, crystalline solid;mp 74-78° C. (EtOH/EtOAc) [lit., (Helferich et al., Just. Lieb. Ann.Chem., 541:1-16 (1939), which is hereby incorporated by reference) mp74-75° C. (EtOH/EtOAc)]; [α]²⁶ _(D)=−22.4 (c 1.63, H₂O) [lit.,(Helferich et al., Just. Lieb. Ann. Chem., 541:1-16 (1939), which ishereby incorporated by reference) [α]¹⁹ _(D)=−26.1 (c 3.0, H₂O)]; ¹H NMR(400 MHz, CD₃OD) δ 3.30 (t, J 8.4 Hz, 1H, H-2), 3.39-3.49 (m, 3H),3.64-3.80 (m, 3H), 3.97 (br d, J_(6,6′) 11.7 Hz, 1H, H-6′), 4.02 (dt,J_(t) 6.5 Hz, J_(d) 11.3 Hz, 1H, —OCHH′—), 4.23 (dt, J_(t) 6.5 Hz, J_(d)11.3 Hz, 1H, —OCHH′—), 4.44 (d, J_(1,2) 7.9 Hz, 1H, H-1). NaSSO₂CH₃ (100mg, 0.75 mmol) was added to a solution of 4c (See FIG. 6) (176 mg, 0.61mmol) in DMF (7 mL) under N₂ and warmed to 50° C. After 15 hours, thesolution was cooled and the solvent removed. The residue was purified byflash chromatography (MeOH:EtOAc, 1:9) to give 1c (See FIG. 6) (144 mg,74%) as a hygroscopic foam; [α]²⁷ _(D)=−15.8 (c 0.88, H₂O); IR (KBr)3400 cm⁻¹ (OH), 1310, 1131 cm⁻¹ (S—SO₂); ¹H NMR (500 MHz, D₂O, COSY) δ3.07 (dd, J_(1,2) 8.1 Hz, J_(2,3) 9.4 Hz, 1H, H-2), 3.16 (dd, J_(3,4)9.0 Hz, J_(4,5) 9.8 Hz, 1H, H-4), 3.24 (ddd, J_(4,5) 9.8 Hz, J_(5,6) 6.0Hz, J_(5,6′) 2.3 Hz, 1H, H-5), 3.27 (t, J 9.0 Hz, 1H, H-3), 3.30-3.33(m, 2H, —CH₂S—), 3.34 (s, 3H, CH₃SO₂—), 3.50 (dd, J_(5,6) 6.0 Hz,J_(6,6′) 12.4 Hz, 1H, H-6), 3.69 (dd, J_(5,6′) 2.3 Hz, J_(6,6′) 12.4 Hz,1H, H-6′), 3.81 (dt, J_(t) 5.8 Hz, J_(d) 11.5 Hz, 1H, —OCHH′—), 4.00(dt, J_(t) 5.7 Hz, J_(d) 11.4 Hz, 1H, —OCHH′—), 4.30 (d. J_(1,2) 8.1 Hz,1H, H-1); ¹³C NMR (50 MHz, D₂O) δ 36.9 (—CH₂S—), 51.0 (CH₃SO₂—), 62.0(—OCH₂—), 69.5, 70.9, 74.3, 76.7, 77.3 (C-2, C-3. C-4, C-5, C-6), 103.7(C-1): HRMS m/z (FAB+): Found 341.0351 (M+Na⁺); C₉H₁₈O₈S₂Na requires341.0341.

[0092] Preparation of 2-(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl)ethyl Methanethiosulfonate (1i).

[0093] BF₃.Et₂O (7.7 mL, 60.7 mmol) was added dropwise over the courseof 15 minutes to a solution of 1,2,3,4,6-penta-O-acetyl-α,β-D-mannose(5d) (See FIG. 7) (4.7 g, 12.1 mmol) and Br(CHI)₂OH (1.05 mL, 14.8 mmol)in CH₂Cl₂ (22 mL) at 0° C. under N₂. After 1 hour, the solution waswarmed to room temperature. After 25 hours, the reaction solution wasadded to ice water (20 mL) and extracted with CH₂Cl₂ (20 mL×2). Theseextracts were combined, washed with water (20 mL), sat. NaHCO₃ (aq., 20mL), water (20 mL), dried (MgSO₄), filtered, and the solvent removed.The residue was crystallized from EtOAc/iso-octane to give 2-bromoethyl2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (4i) (See FIG. 7) (3.52 g,64%). Purification of the resulting mother liquor by flashchromatography (EtOAc:hexane, 1 :3) gave further 4i (See FIG. 7) (320mg, 6%; 70% in total) as a white highly crystalline solid; mp 121-123°C. [lit., (Dahmén et al., “2-Bromoethyl Glycosides—Synthesis andCharacterization,” Carbohydr. Res., 116:303-307 (1983), which is herebyincorporated by reference) 118-119° C. (EtOAc/iso-octane)]; [α]²⁸_(D)=+48.3 (c 1.31, CHCl₃) [lit., (Dahmén et al., “2-BromoethylGlycosides—Synthesis and Characterization,” Carbohydr. Res., 116:303-307(1983), which is hereby incorporated by reference) [α]²³ _(D)=+45 (c0.6, CDCl₃)]; ¹H NMR (200 MHz, CDCl₃) δ 1.99, 2.05, 2.10, 2.16 (s×4,3H×4, Ac×4), 3.52 (t, J 6 Hz, 2H, —CH₂Br), 3.82-4.04 (m, 2H, —OCH₂—),4.09-4.16 (m, 1H, H-5), 4.13 (dd, J_(5,6) 2 Hz, J_(6,6′) 12 Hz, 1H, H-6)4.28 (dd, J_(5,6′) 6 Hz, J_(6,6′) 12 Hz, 1H, H-6′), 4.87 (br s, 1H,H-1), 5.22-5.40 (m, 3H, H-2, H-3, H-4). NaSSO₂CH₃ (230 mg, 1.72 mmol)was added to a solution of 4i (See FIG. 7) (600 mg, 1.32 mmol) in DMF(17 mL) under N₂ and warmed to 55° C. After 20 hours, the solution wascooled and the solvent removed. The residue ,as purified by flashchromatography (EtOAc:hexane, 9:11) and the resulting solidrecrystallized from Et₂O/hexane to give 1i (See FIG. 7) (566 mg, 88%) asa white solid; mp 128-129° C. (Et₂O/hexane); [α]²⁷ _(D)=+53.2 (c 0.92,CHCl₃); IR (KBr) 1739 cm⁻¹ (C═O), 1325, 1129 cm⁻¹ (S—SO₂); ¹H NMR (500MHz, CDCl₃, COSY) δ 1.97, 2.04, 2.09, 2.14 (s×4. 3H×4, Ac×4), 3.37-3.40(m, 2H, —CH₂S—), 3.38 (s, 3H, CH₃SO₂—), 3.79 (dt, J_(d) 10.5 Hz, J_(t)5.8 Hz, 1H, —OCHH′—), 3.98-4.03 (m, 2H, —OCHH′—, H-5), 4.09 (dd, J_(5,6)2.5 Hz, J_(6,6′) 12.5 Hz, 1H, H-6), 4.26 (dd, J_(5,6′) 5.6 Hz, J_(6,6′)12.5 Hz, 1H, H-6′), 4.85 (d, J_(1,2) 0.7 Hz, 1H, H-1), 5.23-5.29 (m, 3H,H-2, H-3, H-4); ¹³C NMR (50 MHz, CDCl₃) δ 20.6, 20.7, 20.8 (CH₃COO—×4),35.7, (—CH₂S—), 50.8 (CH₃SO₂—), 62.5 (—OCH₂—), 66.0, 66.8, 69.0, 69.2,69.3 (C-2, C-3, C-4, C-5, C-6), 97.7 (C-1), 169.7, 169.9, 170.0, 170.6(CH₃ COO—×4); HRMS m/z (FAB+): Found 487.0954 (M+H⁺); C₁₇H₂₇O₁₂S₂requires 487.0944.

[0094] Preparation of 2-(α-D-Mannopyranosyl)ethyl methanethiosulfonate(1d).

[0095] A solution of NaOMe (0.143 M, 0.7 mL) was added to a suspensionof 4i (See FIG. 7) (1 g, 2.2 mmol) in MeOH (10 mL) under N₂. After 3hours, the resulting solution was passed through a Dowex 50W(H⁺) plug(2×1 cm, eluant MeOH) and the solvent removed. The residue was purifiedby flash chromatography (MeOH:EtOAc, 2:25) to give 2-bromoethylα-D-mannopyranoside 4d (See FIG. 7) (The use of 4d as a reactant hasbeen described previously, although no details of preparation orcharacterization were given. (U.S. Pat. No. 4,918,009 to Nilsson, whichis hereby incorporated by reference)) (606 mg, 96%) as a white foam;[α]²⁶ _(D)=+50.7 (c 0.91, H₂O); IR (KBr) 3417 cm⁻¹ (OH); ¹H NMR (500MHz. D₂O. COSY) δ 3.38-3.44 (m, 3H, H-4, —CH₂Br), 3.50-3.55 (m, 2H, H-5,H-6), 3.60 (dd, J_(2,3) 3.5 Hz, J_(3,4) 9.7 Hz, 1H, H-3), 3.66 (dd,J_(5,6′) 4.6 Hz, J_(6,6′) 11.2 Hz, 1H, H-6′), 3.68 (ddd, J 4.6 Hz, J 5.4Hz, J 11.7 Hz, 1H, —OCHH′—), 3.76 (dd, J_(1,2) 1.8 Hz, J_(2,3) 3.5 Hz,1H, H-2), 3.81 (ddd, J 5.1 Hz, J 6.5 Hz, J 11.7, 1H, —OCHH′—), 4.71 (d,J_(1,2) 1.8 Hz, 1H, H-1); ^(—)C NMR (100 MHz, D₂O) δ 32.1 (—CH₂Br), 61.7(—OCH₂—), 67.5, 68.4, 70.7, 71.3, 73.8 (C-2, C-3, C-4, C-5, C-6), 100.5(C-1); HRMS m/z (FAB+): Found 308.9985 (M+Na⁺); C₈H₁₅O₆ ⁷⁹BrNa requires308.9950. NaSSO₂CH₃ (150 mg, 1.12 mmol) was added to a solution of 4d(See FIG. 7) (245 mg, 0.85 mmol) in DMF (10 mL) under N₂ and warmed to50° C. After 16 hours, the solution was cooled and the solvent removed.The residue was purified by flash chromatography (MeOH:EtOAc, 1:9) togive 1d (See FIG. 7) (217 mg, 80%) as a hygroscopic foam; [α]²⁹_(D)=+58.0 (c 1.34, H₂O); IR (KBr) 3441 cm⁻¹ (OH), 1314, 1132 cm⁻¹(S—SO₂); ¹H NMR (500 MHz, D₂O) δ 3.31 (t, J 5.8 Hz, 2H, —CH₂S—), 3.35(s, 3H, CH₃SO₂—), 3.45 (t, J 9.6 Hz, 1H, H-4), 3.49 (ddd, J_(4,5) 9.8Hz, J_(5,6) 5.8 Hz, J_(5,6′) 1.9 Hz, 1H, H-5), 3.55 (dd, J_(5,6) 5.8 Hz,J_(6,6′) 12.1 Hz, 1H, H-6), 3.60 (dd, J_(2,3) 3.4 Hz, J_(3,4) 9.0 Hz,1H, H-3), 3.66 (dt, J_(d) 10.7 Hz, J_(t) 5.7 Hz, 1H, —OCHH′—), 3.69 (dd,J_(5,6) 1.9 Hz, J_(6,6′) 12.1 Hz, 1H, H-6′), 3.77 (dd, J_(1,2) 1.6 Hz,J_(2,3) 3.4 Hz, 1H, H-2), 3.83 (dt, J_(d) 11.0 Hz, J_(t) 5.9 Hz, 1H,—OCHH′—), 4.72 (d, J_(1,2) 1.6 Hz, 1H, H-l); ¹³C NMR (125 MHz, D₂O) δ36.7 (—CH₂S—), 50.7 (CH₃SO₂—), 61.9 (—OCH₂—), 66.7, 67.7, 70.9, 71.5,74.0 (C-2, C-3, C-4, C-5, C-6), 100.8 (C-1); HRMS m/z (FAB+): Found319.0528 (M+H⁻); C₉H₁₉O₈S₂ requires 319.0521.

[0096] Preparation of 2-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl)ethyl Methanethiosulfonate (1j).

[0097] BF₃.Et₂O (8.5 mL, 67.0 mmol) was added dropwise to a solution ofof 1,2,3,4,6-penta-O-acetyl-α,β-D-galactose (5e) (See FIG. 7) (5.1 g,13.1 mmol) and Br(CH₂)₂OH (1.15 mL, 16.2 mmol) in CH₂Cl₂ (24 mL) at 0°C. under N₂. After 1 hour, the solution was warmed to room temperature.After 24 hours, the reaction solution was added to ice water (20 mL) andextracted with CH₂Cl₂ (30 mL×3). These extracts were combined, washedwith water (20 mL), sat. NaHCO₃ (aq., 20 mL), water (20 mL), dried(MgSO₄), filt Vögtle ered, and the solvent removed. The residue purifiedby flash chromatography (EtOAc:hexane, 1:3) to give 2-bromoethyl2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (4j) (See FIG. 7) (4.01 g,67%) as a white solid; mp 116-117° C. (EtOAc/hexane) [lit., (Coles etal., J. Am. Chem. Soc., 60:1020-1022 (1938), which is herebyincorporated by reference) 111° C.; lit., (Dahmén et al., “2-BromoethylGlycosides—Synthesis and Characterization,” Carbohydr. Res., 116:303-307(1983), which is hereby incorporated by reference) 114-116° C.(EtOAc/light pet. ether)]; [α]²⁷ _(D)=−3.8 (c 0.81, CHCl₃) [lit.,(Dahmén et al., “2-Bromoethyl Glycosides—Synthesis andCharacterization,” Carbohydr. Res., 116:303-307 (1983), which is herebyincorporated by reference) [a]23D=-5 (c 1.4, CDCl₃)]; ¹H NMR (200 MHz,CDCl₃) δ 1.98, 2.05, 2.08, 2.15 (s×4, 3H×4, Ac×4), 3.43-3.50 (m, 2H),3.75-3.95 (m, 2H), 4.12-4.24 (m, 3H), 4.53 (d, J_(1.2) 8 Hz, 1H, H-1),5.02 (dd, J_(2,3) 11 Hz, J_(2,3) 3 Hz, 1H, H-3), 5.23 (dd, J_(1,2) 8 Hz,J_(2,3) 11 Hz, 1H, H-2), 5.40 (br d, J_(3,4) 3 Hz, 1H, H-4). NaSSO₂CH₃(85 mg, 0.63 mmol) was added to a solution of 4j (See FIG. 7) (223 mg,0.49 mmol) in DMF (6 mL) under N₂ and warmed to 55° C. After 30 hours,the solution was cooled and the solvent removed. The residue waspurified by flash chromatography (EtOAc:hexane, 1:1) to give 1j (SeeFIG. 7) (198 mg, 83%) as a white foam; [α]²⁷ _(D)=+9.1 (c 1.41. CHCl₃);IR (film) 1747 cm⁻¹ (C═O), 1320, 1133 cm⁻¹ (S—SO₂); ¹H NMR (500 MHz,CDCl₃) δ 1.98, 2.05, 2.09, 2.15 (s×4, 3H×4, Ac×4), 3.35 (s, 3H,CH₃SO₂—), 3.35-3.38 (m, 2H, —CH₂S—), 3.84 (ddd, J 6.1 Hz, J 7.1 Hz, J10.5 Hz, 1H, —OCHH′—), 3.92 (td, J_(4,5) 1.1 Hz, J_(t) 6.6 Hz, 1H, H-5),4.10-4.21 (m, 3H, H-6, H-6′, —OCHH′—), 4.52 (d, J_(1,2) 8.0 Hz, 1H,H-1), 5.01 (dd, J_(2,3) 10.3 Hz, J_(3,4) 3.5 Hz, 1H, H-3),5.20 (dd,J_(1,2) 8.0 Hz, J_(2,3) 10.3 Hz, 1H, H-2), 5.40 (dd, J_(3,4) 3.5 Hz,J_(4,5) 1.1 Hz, 1H, H-4); ¹³C NMR (100 MHz, CDCl₃) δ 20.6,20.7, 20.8(CH₃COO—×4), 36.1, (—CH₂S—), 50.6 (CH₃SO₂—), 61.2 (—OCH₂—), 67.0, 68.3,68.5, 70.8, 71.0 (C-2, C-3, C-4, C-5, C-6), 101.3 (C-1), 169.5, 170.0,170.1, 170.4 (CH₃COO—×4); HRMS m/z (FAB+): Found 487.0936 (M+H⁺);C₁₇H₂₇O₁₂S₂ requires 487.0944.

[0098] Preparation of 2-(β-D-Galactopyranosyl)ethyl Methanethiosulfonate(1e).

[0099] A solution of NaOMe (0.104 M, 0.8 mL) was added to a solution of4j (See FIG. 7) (778 mg. 1.71 mmol) in MeOH (10 mL) under N₂. After 4hours, the reaction solution was passed through a Dowex 50W(H⁺) plug(3×1 cm, eluant MeOH) and the solvent removed to give 2-bromoethylβ-D-galactopyranoside (4e) (See FIG. 7) (450 mg, 92%) (The synthesis ofunstable 4e has been described previously. Dahmén et al., “2-BromoethylGlycosides 4.2-Bromoethyl Glycosides in Glycoside Synthesis—Preparationof Glycoproteins Containing Alpha-L-Fuc-(1->2)-D-Gal andBeta-D-Gal-(1->4)-D-Glcnac,” Carbohydr. Res., 125:237-245 (1984), whichis hereby incorporated by reference) as a white solid which was useddirectly in the next step. NaSSO₂CH₃ (180 mg, 1.34 mmol) was added to asolution 4e (See FIG. 7) (290 mg, 1.01 mmol) in DMF (12 mL) under N₂ andwarmed to 50° C. After 15 hours, the solution was cooled-and the solventremoved. The residue was purified by flash chromatography (MeOH:EtOAc,1:9) to give 1e (See FIG. 7) (229 mg, 71%) as a white foam; [α]²⁷^(D)=+2.9 (c 0.58, H₂O); IR (film) 3358 cm⁻¹ (br, O—H), 1306, 1120 cm⁻¹(S—SO₂); ¹H NMR (500 MHz, D₂O, COSY) δ 3.29-3.33 (m, 2H, —CH₂S—), 3.30(dd, J_(1,2) 7.7 Hz, J_(2,3) 10.0 Hz, 1H, H-2), 3.35 (s, 3H, CH₃SO₂—),3.43 (dd, J_(2,3) 10.0 Hz, J_(3,4) 3.6 Hz, 1 H, H-3), 3.48 (ddd, J_(4,5)0.9 Hz, J_(5,6) 4.3 Hz, J_(5,6′) 7.9 Hz, 1H, H-5), 3.52 (dd J_(5,6) 4.3Hz, J_(6,6′) 11.7 Hz, 1H, H-6),3.57(dd, J_(5,6′) 7.9 Hz, J_(6,6′) 11.7Hz, 1H, H-6′) 3.70 (dd, J_(3,4) 3.6 Hz, J_(4,5) 0.9 Hz, 1H, H-4), 3.80(dt, J_(d) 11.2 Hz, J_(t) 6.1 Hz, 1H, —OCHH′—), 4.01 (dt, J_(d) 11.4 Hz,J_(t) 5.8 Hz, 1H, —OCHH′—), 4.24 (d, J_(1,2) 7.7 Hz, 1H, H-1); ¹³C NMR(100 MHz, D₂O) δ 36.7 (—CH₂S—), 50.8 (CH₃SO₂—), 61.9 (—OCH₂—), 69.2,69.6, 71.7. 73.7, 76.2 (C-2, C-3, C-4, C-5, C-6), 104.0 (C-1); HRMS m/z(FAB+): Found 319.0523 (M+H⁺); C₉H₁₉O₈S₂ requires 319.0521.

[0100] Preparation of2-(2,3,6-Tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-β-D-glucopyranosyl)ethylMethanethiosulfonate (1k).

[0101] BF₃.Et₂O (4.0 mL, 31.5 mmol) was added dropwise to a solution of1,2,3,6-tetra-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-β-D-glucopyranoside (5f) (See FIG. 7) (5g, 7.4 mmol) and Br(CH₂)₂OH (0.65 mL, 9.2 mmol) in CH₂Cl₂ (15 mL) at 0°C. under N₂. After 1 hour, the solution was warmed to room temperature.After 20 hours, the reaction solution was added to ice water (15 mL) andextracted with CH₂Cl₂ (20 mL×2). These extracts were combined, washedwith water (20 mL), sat. NaHCO₃ (aq., 20 mL), water (20 mL), dried(MgSO₄), filtered, and the solvent removed. The residue was purified byflash chromatography (EtOAc:hexane, 1:1) to give 2-bromoethyl2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-β-D-glucopyranoside(4k) (See FIG. 7) (2.94 g, 53%) as a white foam; [α]²⁷ _(D)=−7.8 (c1.28, CHCl₃) [lit., (Dahmén et al., “2-Bromoethyl Glycosides—Synthesisand Characterization,” Carbohydr. Res., 116:303-307 (1983), which ishereby incorporated by reference) [α]²³ _(D)=−11 (c 1.3, CHCl₃)]; ¹H NMR(500 MHz, CDCl₃, COSY) δ 1.94, 2.02, 2.02 (s×3, 3H×3, Ac×3), 2.04 (s,6H, Ac×2), 2.10, 2.13 (s×2, 3H×2, Ac×2), 3.38-3.46 (m, 2H, —CH₂Br), 3.59(ddd, J_(5′,6′″) 2.2 Hz, J 4.9 Hz, J 9.9 Hz, 1H, H-5′), 3.75-3.80 (m,2H, H-4′, —OCHH′—), 3.85 (td, J_(4,5) 1.1 Hz, J_(t) 6.9 Hz, 1H, H-5),4.03-4.12 (m, 4H, H-6, H-6′, H-6″, —OCHH′—), 4.45 (d, J_(1,2) 7.8 Hz,1H, H-1), 4.48 (dd, J_(5′,6′″) 2.2 Hz, J_(6″,6′″) 12.1 Hz, 1H, H-6′″),4.50 (d, J_(1′,2′) 7.9 Hz, 1H, H-1′), 4.89 (dd, J_(1′,2′) 7.9 Hz,J_(2′,3′) 9.6 Hz, 1H, H-2′), 4.92 (dd, J_(2,3) 10.5 Hz, J_(3,4) 3.4 Hz,1H, H-3), 5.08 (dd, J_(1,2) 7.8 Hz, J_(2,3) 10.5 Hz, 1H, H-2), 5.18 (t,J 9.6 Hz, 1H, H-3′), 5.32 (dd, J_(3,4) 3.4 Hz, J_(4,5) 1.1 Hz, 1H, H-4).NaSSO₂CH₃ (87.mg, 0.65 mmol) was added to a solution of 4k (See FIG. 7)(357 mg, 0.48 mmol) in DMF (6 mL) under N₂ and warmed to 50° C. After 22hours, the solution was cooled and the solvent removed. The residue waspurified by flash chromatography (EtOAc:hexane, 11:9) to give 1k (SeeFIG. 7) (327 mg, 88%) as a white foam; [α]²⁷ _(D)=−3.7 (c 1.0, CHCl₃);IR (KBr) 1751 cm⁻¹ (C═O), 1323, 1134 cm⁻¹ (S—SO₂); ¹H NMR (500 MHz,CDCl₃, COSY) δ 1.94, 2.02, 2.02, 2.04, 2.04, 2.11, 2.13 (s×7, 3H×7,Ac×7), 3.29-3.40 (m, 2H, -CH₂S-), 3.32 (s. 3H, CH₃SO₂—), 3.59 (ddd,J_(4′,5′) 9.9 Hz, J_(5′,6″) 4.9 Hz, J_(5′,6′″) 2.2 Hz, 1H, H-5′), 3.77(t, J 9.5 Hz, 1H, H-4′), 3.79-3.86 (m, 2H, H-5, —OCHH′—), 4.03-4.13 (m,4H, H-6, H-6′, H-6″. —OCHH′—), 4.46 (d, J_(1,2) 7.8 Hz, 1H, H-1), 4.50(d, J_(1′,2′) 8.0 Hz, 1H, H-1′), 4.52 (dd, J_(5′,6′) 62.2 Hz, J_(6″,6′″)11.9 Hz, 1H, H-6′″), 4.87 (dd, J_(1′,2′) 8.0 Hz, J_(2′,3′) 9.6 Hz, 1H,H-2′), 4.93 (dd, J_(2,3) 10.5 Hz, J_(3,4) 3.5 Hz, 1H, H-3), 5.08 (dd,J_(1,2) 7.8 Hz, J_(2,3) 10.5 Hz, 1H, H-2), 5.17 (t, J 9.3 Hz, 1H, H-3′),5.32 (dd, J_(3,4) 3.5 Hz, J_(4,5) 1.0 Hz, 1H, H-4); ¹³C NMR (125 MHz,CDCl₃, DEPT) δ 20.5, 20.7, 20.8, 20.9 (q×4, CH₃COO—×7), 36.0 (t,—CH₂S—), 50.6 (q, CH₃SO₂—), 60.7, 61.6, 68.5 (t×3, —OCH₂—, C-6, C-6′),66.5, 69.0, 70.6, 70.9, 71.3, 72.6, 72.8, 76.0 (d×8, C-2, C-3, C-4, C-5,C-2′, C-3′, C-4′, C-5′), 100.7, 101.1 (d×2, C-1, C-1′), 169.1, 169.7,169.7, 170.1, 170.2, 170.3, 170.4 (s×7, CH₃ COO—×7); HRMS m/z (FAB+):Found 775.1793 (M+H⁺); C₂₉H₄₃O₂₀S₂ requires 775.1789.

[0102] Preparation of 2-(4-O-β-D-Galactopyranosyl-β-D-glucopyranosyl)ethyl Methanethiosulfonate(1f).

[0103] A solution of NaOMe (0.1 M, 0.6 mL) was added to a solution of 4k(See FIG. 7) (877 mg, 1.18 mmol) in MeOH (6 mL) under N₂. After 3 hours,the reaction solution was passed through a Dowex 50W(H⁺) plug (4×1 cm,eluant MeOH) and the solvent removed to give 2-bromoethyl4-O-β-D-galactopyranosyl-β-D-glucopyranoside (4f) (See FIG. 7) (476 mg,90%) as a white foam which was used directly in the next step. NaSSO₂CH₃(185 mg, 1.38 mmol) was added to a solution of 4f (See FIG. 7) (476 mg,1.06 mmol) in DMF (24 mL) under N₂ and warmed to 50° C. After 21 hours,the solution was cooled and the solvent removed. The residue waspurified by flash chromatography (CHCl₃:MeOH:AcOH:H₂O, 60:30:3:5) togive 1f (See FIG. 7) (346 mg, 68%) as a hygroscopic foam; [α]²⁸_(D)=+1.5 (c 1.66, H₂O); IR (KBr) 3416 cm⁻¹ (br, OH), 1311, 1131 cm⁻¹(S—SO₂), ¹H NMR (400 MHz, D₂O, COSY) δ 3.10-3.13 (m, 1H, H-2), 3.30 (t,J 6.0 Hz, 2H —CH₂S—), 3.31 (dd, J_(1′,2) 7.8 Hz, J_(2′,3) 10.3 Hz, 1H,H-2′), 3.34 (s, 3H, CH₃SO₂—), 3.38-3.54(m, 5H), 3.44 (dd, J_(2′,3′) 10.3Hz, J_(3′,4′) 3.3 Hz, 1H, H-3′), 3.57 (dd, J 8.4 Hz, J 11.4 Hz, 1H),3.59(dd, J 4.9 Hz, J 7.3 Hz, 1H), 3.70 (br d, J_(3′,4′) 3.3 Hz, 1H, H-4′),3.77 (dd, J 1.0 Hz, J 11.5 Hz, 1H), 3.79-3.83 (m, 1H, —OCHH′—),3.97-4.02 (m, 1H, —OCHH′—), 4.22 (d, J_(1,2) 7.8 Hz, 1H, H-1′), 4.33 (d,J_(1,2) 7.8 Hz, 1H, H-1); ¹³C NMR (125 MHz, D₂O) δ 36.7 (—CH₂S—), 50.8(CH₃SO₂—), 61.0, 62.1, 69.4, 69.6, 71.9, 73.5, 73.7, 75.3, 75.9, 76.4,79.3 (—OCH₂—, C-2, C-3, C-4, C-5, C-6, C-2′, C-3′, C-4′, C-5′, C-6′),103.3, 103.9 (C-1, C-1′); HRMS m/z (FAB+): Found 503.0886 (M+Na⁺);C₁₅H₂₈O₃S₂Na requires 503.0869.

Example 2 General Procedure for Modification of Subtilisin Bacilluslentus (“SBL”) Mutants Stored as Flash-Frozen Solutions

[0104] A 1.25 mL frozen aliquot of the mutant enzyme (SBL-N62C, -L217C,or -S166C) containing approximately 25 mg of enzyme was thawed and addedto 1.25 mL of Modifying Buffer (see below) in a polypropylene test-tube.To this solution was added 100 uL of a 0.2 M glyco-MTS reagent solution(1a,g-k in MeCN, 1b-f in water (See FIG. 8)). The mixture was sealed,vortexed, and placed on an end-over-end rotator at room temperature.When the modification was complete (determined by a specific activityassay, using succinyl-AlaAlaProPhe-p-nitroanilide [ε₄₁₀=8800 M⁻¹ cm⁻¹](Bonneau et al., “Alteration of the Specificity of Subtilisin BPN′ bySite-Directed Mutagenesis in its S1 and S1′ Binding-Sites,” J. Am. Chem.Soc., 119:1026-1030 (1991), which is hereby incorporated by reference)as substrate in 0.1 M Tris-HCl buffer containing 0.005% Tween 80, 1%DMSO, pH 8.6 showing constant activity and titration with Ellman'sreagent (ε₄₁₂=13600 M⁻¹ cm⁻¹) (Ellman et al., Biochem. Pharmacol.,7:88-95 (1961), which is hereby incorporated by reference) showing nofree thiol present in solution), a further 50 μL of the modifyingreagent solution was added and the mixture placed back on theend-over-end rotator for a further 10 minutes. The reaction was pouredonto a pre-packed, pre-equilibrated G-25 Sephadex® PD10 column andeluted with 3.5 mL Quench Buffer (see below). The eluant was dialysed at4° C. against 10 mM MES, 1 mM CaCl₂ pH 5.8 (2×1L, 2×45 minutes). Theresulting dialysate was flash frozen in liquid nitrogen and stored at−18° C. Modifying Buffer: pH 9.5: 140 mM CHES, 2 mM CaCl₂ pH 7.5: 140 mMHEPES.2 mM CaCl₂ pH 6.5: 140 mM MES, 2 mM CaCl₂ pH 5.5: 140 mM MES, 2 mMCaCl₂ Quench Buffer: Reactions at pH 7.5-9.5: 5 mM MES 1 mM CaCl₂ pH 6.5Reactions at pH 5.5: 5 mM MES 1 mM CaCl₂ pH 5.5

[0105] The free thiol content of all chemically modified mutant enzymes(“CMMs”), was determined spectrophotometrically by titration withEllman's reagent (Ellman et al., Biochem. Pharmacol., 7:88-95 (1961),which is hereby incorporated by reference) in phosphate buffer 0.25 M,pH 8.0. In all cases, no free thiol was detected. Modified enzymes wereanalyzed by nondenaturing gradient (8-25%) gels at pH 4.2, run towardsthe cathode, on the Pharmacia Phast-system and appeared as a singleband. Prior to ES-MS analysis, CMMs were purified by FPLC (BioRad,Biologic System, Hercules, Calif.) on a Source 15 RPC matrix (17-0727-20from Pharmacia, Bridgewater, N.J.) with 5% acetonitrile, 0.01% TFA asthe running buffer and eluted with 80% acetonitrile, 0.01% TFA in a onestep gradient. MS m/z (ES-MS): N62C-S-a (See FIG. 8) calculated 27049,found 27051; N62C-S-b (See FIG. 8) calculated 26925, found 26928;N62C-S-c (See FIG. 8) calculated 26925, found 26928; N62C-S-d (See FIG.8) calculated 26925, found 26925; N62C-S-e (See FIG. 8) calculated26925, found 26925; N62C-S-f (See FIG. 8) calculated 27087, found 27087;N62C-S-g (See FIG. 8) calculated 27093, found 27096;N62C-S-Et-,-Glc(Ac)₂ calculated 27009, found 27015; N62C-S-Et-P-Glc(Ac)₃calculated 27051, found 27053; N62C-S-i (See FIG. 8) calculated 27093,found 27098; N62C-S-Et-β-Gal(Ac)₃ calculated 27051, found 27051;N62C-S-k (See FIG. 8) calculated 27381, found 27386; L217C-S-β-Glccalculated 26882, found 26879; L217C-S-β-Glc(Ac)₂ calculated 26966,found 26962; L217C-S-β-Glc(Ac)₃ calculated 27008, found 27006; L217C-S-b(See FIG. 8) calculated 26926, found 26928; L217C-S-c (See FIG. 8)calculated 26926, found 26925. L217C-S-d (See FIG. 8) calculated 26926,found 26925; L217C-S-e (See FIG. 8) calculated 26926, found 26928;L217C-S-f (See FIG. 8) calculated 27088, found 27087;L217C-S-Et-α-Glc(Ac)₂ calculated 27010, found 27012;L217C-S-Et-β-Glc(Ac)₃ calculated 27052, found 27056;L217C-S-Et-α-Man(Ac)₃ calculated 27052, found 27056;L217C-S-Et-β-Gal(Ac)₃ calculated 27052, found 27053; L217C-S-Et-Lac(Ac)₆calculated 27340, found 27342; S166C-S-a (See FIG. 8) calculated 27076,found 27080; S166C-S-b (See FIG. 8) calculated 26952, found 26955;S166C-S-c (See FIG. 8) calculated 26952, found 26950; S166C-S-d (SeeFIG. 8) calculated 26952, found 26952; S166C-S-e (See FIG. 8) calculated26952, found 26952; S166C-S-f (See FIG. 8) calculated 27114, found27112; S166C-S-Et-α-Glc(Ac)₃ calculated 27078, found 27078;S166C-S-Et-β-Glc(Ac)₂ calculated 27036, found 27040;S166C-S-Et-β-Glc(Ac)₃ (major) with S166C-S-h (See FIG. 8) (minor) andS166C-S-Et-β-Glc(Ac)₂ (minor) calculated 27078 (major), 27120 (minor),27036 (minor), found 27081 (major), 27121 (minor), 27036 (minor);S166C-S-Et-α-Man(Ac)₃ calculated 27078, found 27085;S166C-S-Et-β-Gal(Ac)₃ calculated 27078, found 27079; S166C-S-Et-Lac(Ac)₅calculated 27324, found 27331.

Example 3 General Procedure for Modification of SBL Mutants Stored asLyophilized Powders

[0106] This procedure was only used with S156C, which is stored as alyophilized powder to prevent dimerization. Into a polypropylene testtube was weighed about 25-30 mg of lyophilized S156C. This was dissolvedin the following modifying buffers (2.5 mL): pH 9.5:   70 mM CHES, 2 mMCaCl₂ pH 7.5:   70 mM HEPES, 2 mM CaCl₂ pH 6.5:   70 mM MES, 2 mM CaCl₂pH 5.5:   70 mM MES, 2 mM CaCl₂

[0107] Glyco-MTS reagent was added and the reaction then proceeded asfor the other mutants, using the appropriate quench buffer. MS m/z(ES-MS): S156C-S-a (See FIG. 8) calculated 27076, found 27079; S156C-S-b(See FIG. 8) calculated 26952, found 26955; S156C-S-c (See FIG. 8)calculated 26952, found 26952; S156C-S-d (See FIG. 8) calculated 26952,found 26952; S156C-S-e (See FIG. 8) calculated 26952, found 26952;S156C-S-f (See FIG. 8) calculated 27114, found 27115; S156C-S-g (SeeFIG. 8) calculated 27120, found 27123; S156C-S-h (See FIG. 8) calculated27120, found 27122; S156C-S-i (See FIG. 8) calculated 27120, found27123; S156C-S-j (See FIG. 8) calculated 27120, found 27120; S156C-S-k(See FIG. 8) calculated 27408, found 27411.

Example 4 Contents of Acetylated Glyco-CMM Libraries

[0108] The levels of acetylation of glyco-CMMs after modification of SBLcysteine mutants with 1a,g-k (See FIG. 8) at various pH levels weredetermined and are set forth in Tables 1 and 2, below. TABLE 1 Levels ofAcetylation of Glyco-CMMs after Modification of SBL Cysteine Mutantswith 1a at various pH^(a) Reagent 1a 1a 1a Enzyme pH 9.5 pH 7.5 pH 5.5N62C 4 — 4 S156C 4 — 4 S166C 4 — 4 L217C 0 2^(b) 3^(b)

[0109] TABLE 2 Levels of Acetylation of Glyco-CMMs after Modification ofSBL Cysteine Mutants with 1a,g-k at pH 5.5^(a) Reagent Enzyme 1a 1g 1h1i 1j 1k N62C 4 4 3^(b) 4  3^(b) 7  S156C 4 4 4  4  4  7  S166C 4 33^(c), 4^(d), 2^(d) 3^(b) 3^(b) 5^(b) L217C  3^(b)  2^(b) 3^(b) 3^(b)3^(b) 6^(b)

Example 5 Incubation of L217C-S-β-Glc(Ac)₃ at pH 9.5

[0110] The general procedure for modification of SBL mutants stored asflash-frozen solutions was used to incubate 1.26 mg ofL217C-S-β-Glc(Ac)₃ as a 0.5 mL aliquot in the absence of MTS reagent for2 hours to give L217C-S-β-Glc as the sole product. MS m/z (ES-MS):L217C-S-β-Glc calculated 26882, found 26885.

Example 6 Active Site Titrations

[0111] The active enzyme concentration was determined as previouslydescribed (Hsia et al., “Active-Site Titration of Serine Proteases Usinga Fluoride-Ion Selective Electrode and Sulfonyl Fluoride Inhibitors,”Anal. Biochem., 242:221-227 (1996), which is hereby incorporated byreference) by monitoring fluoride release upon enzyme reaction withα-toluenesulfonyl fluoride (PMSF) as measured by a fluoride ionsensitive electrode (Orion Research 96-09). The active enzymeconcentration determined in this way was used to calculate k_(cat)values for each CMM.

Example 7 Kinetic Measurements

[0112] Michaelis-Menten constants were measured at 25(±0.2)° C. by curvefitting (GraFit® 3.03, Erithacus Software Ltd., Staines, Middlesex, UK)of the initial rate data determined at nine concentrations (0.125 mM-3.0mM) of succinyl-AAPF-pNA substrate in 0.1 M Tris-HCl buffer containing0.005% Tween 80, 1% dimethylsufoxide (“DMSO”), pH 8.6 (ε₁₁₀=8800 M⁻¹cm⁻¹) (Bonneau et al., J. Am. Chem. Soc., 119:1026-1030 (1991), which ishereby incorporated by reference).

Example 8 Controlled Site Selective Glycosylation of Proteins by aCombined Site-Directed Mutagenesis and Chemical Modification Approach

[0113] Four SBL sites at different locations and of differentcharacteristics were selected for mutation to cysteine in order toprovide a broad test of the glycosylation methodology. S156 of theS,-pocket (Nomenclature of Schechter; Berger, Biochem. Biophys. Res.Commun., 27:157-162 (1967), which is hereby incorporated by reference)is a surface-exposed residue that permits the introduction ofexternally-disposed glycans mirroring those found naturally inglycoproteins (Molecular Glycobiology, Fukuda et al., Eds., OxfordUniversity, Oxford (1994), which is hereby incorporated by reference).In contrast, N62 in the S₂ pocket, S166 in the S₁ pocket, and L217 inthe S₁′ pocket have side chains which are internally oriented and testthe applicability of the method for introducing sugars at hinderedlocations. Broad applicability with respect to the sugar moiety wasevaluated by using the representative series of protected anddeprotected, mono- and disaccharide methanethiosulfonates (“MTS”) 1a-k(see FIG. 8). These were prepared from their parent carbohydrates ingood to excellent yields (FIG. 6 (Reagents and Conditions: (i) Ac₂O, pythen HBr, AcOH; (ii) NaSSO₂CH₃, EtOH, 90° C.; (iii) Br(CH₂)₂OH, BF₃.Et₂Othen Ac₂O, py; (iv) NaSSO₂CH₃, DMF, 50° C.; (v) NaOMe, MeOH; (vi) Ac₂O,py then Br(CH₂)₂OH, BF₃.Et₂O, DCM) and 7 (Reagents and Conditions: (i)Ac₂O, py, 92% for 5d, 99% for 5e; Ac₂O, NaOAc, 82% for 5f; (ii)Br(CH₂)₂OH, BF₃.Et₂O, DCM, 70% for 4i, 67% for 4j, 53% for 4k; (iii)NaOMe, MeOH, 96% for 4d, 92% for 4e, 90% for 4f; (iv) NaSSO₂CH₃, DMF,50° C., 80% for 1d, 71% for 1e, 68% for 1f, 88% for 1i, 83% for 1j, 88%for 1k)). Two types of glycosylating reagents, the anomericmethanethiosulfonate 1a and the ethyl-tethered methanethiosulfonates1b,c,g,h, were prepared from D-glucose (2a, FIG. 6). The preparation ofthese reagents in fully protected 1a,g,h and deprotected 1b,c formsallowed the effects of increased steric bulk and hydrophobicity to beassessed. Untethered MTS reagent 1a was readily prepared fromacetobromoglucose (3) using NaSSO₂CH₃ as shown in FIG. 6 (Prepared fromD-glucose according to Scheurer et al., J. Am. Chem. Soc., 76:3224(1954), which is hereby incorporated by reference). For the preparationof 1b,g, an α-linked ethyl tether was introduced using Fischerglycosidation of D-glucose (2) with 2-bromoethanol. Treatment of thetetraacetylbromide 4g with NaSSO₂CH₃ allowed the preparation of theperacetylated α-gluco-MTS 1g in an excellent 90% yield. Zemplendeacylation (Zemplén et al., Ber. Dtsch. Chem. Ges., 56:1705-1710(1923), which is hereby incorporated by reference) of bromide 4g andsubsequent displacement of bromide by methanethiosulfonate ion proceededsmoothly to yield the fully deprotected α-gluco-MTS 1b in 69% yield. Theβ-D-gluco-MTS reagents 1c and 1h, which are epimeric at C-1 relative to1b and 1g, respectively, were prepared from the correspondingperacetylated P-bromide 4h. The preparation of 4h took advantage ofwell-defined methodology utilizing Lewis acid catalyzed displacement ofanomeric acetates by alcohols (Dahmén et al., “2-BromoethylGlycosides—Synthesis and Characterization,” Carbohydr. Res., 116:303-307(1983), which is hereby incorporated by reference). The protectedbromide 4h was elaborated to the corresponding peracetylated (1h) anddeprotected (1c) β-gluco-MTS reagents in an essentially identical mannerto that used for the epimeric α-gluco-MTS reagents. Thus, usingNaSSO₂CH₃, 4h gave 1h in 78% yield and, following deprotection, 4c(Helferich et al., Just. Lieb. Ann. Chem., 541:1-16 (1939), which ishereby incorporated by reference) afforded 1c in 68% yield from 4h.Parallel routes allowed similarly efficient access to the α-D-manno-MTSreagents 1d and 1i, which are epimeric at C-2 relative to 1b and 1g,respectively, and the β-D-galacto-MTS reagents 1e and 1j, epimeric atC-4 relative to 1c and 1h, respectively (FIG. 7). The ready adaptabilityof this method to oligosaccharides was illustrated by the preparation ofthe peracetylated (1k) and fully deprotected (1f) disaccharide lacto-MTSreagents, in good overall yields from lactose (2f) of 38% and 27%respectively without cleavage of the interresidue bond.

Example 9 Site Specific Glycosylation

[0114] The glyco-MTS reagents la-k (See FIG. 8) were reacted with thechosen cysteine mutants SBL-N62C, -S156C, -S166C and -L217C in aqueousbuffer under conditions described previously (Stabile et al., Bioorg.Med. Chem. Lett., 6:2501-2512 (1996); Berglund et al., J. Am. Chem.Soc., 119:5265-5266 (1997); DeSantis et al. Biochem., 37:5968-5973(1998), which are hereby incorporated by reference). These reactionswere rapid and quantitative, as judged by monitoring of changes inspecific activity and by titration of free thiols with Ellman's reagent(Ellman et al., Biochem. Pharmacol., 7:88-95 (1961), which is herebyincorporated by reference). The glycosylated chemically modified mutants(CMMs) were purified by size-exclusion chromatography and dialysis, andtheir structures were confirmed by rigorous ES-MS analyses (±7 Da) asshown in Table 3 below: TABLE 3 Properties and Kinetic Parameters^(a) ofModified Enzymes Reactant MTS k_(cal)/K_(M) Entry Enzyme Pocket ReagentReacn. pH Products(s) k_(cal) (s⁻¹) K_(M) (mM) (s⁻¹mM⁻¹) 1 SBL-WT — — —— 153 ± 4  0.73 ± 0.05 209 ± 15 2 N62C S₂ — — — 174 ± 9  1.90 ± 0.20  92± 11 3 1a 9.5 N62C-S-a^(b) 67.9 ± 3.5 0.52 ± 0.07 130.6 ± 18.8 4 1b 6.5N62C-S-b^(b) 135.3 ± 3.5  0.94 ± 0.05 143.9 ± 8.5  5 1c 6.5 N62C-S-c^(b)132.7 ± 4.0  1.25 ± 0.08 106.2 ± 7.5  6 1d 6.5 N62C-S-d^(b) 132.9 ± 3.1 1.04 ± 0.05 127.8 ± 6.8  7 1e 6.5 N62C-S-e^(b) 119.3 ± 3.6  0.99 ± 0.07120.5 ± 9.3  8 1f 6.5 N62C-S-f^(b) 129.8 ± 2.4  1.04 ± 0.04 124.8 ± 5.3 9 1g 5.5 N62C-S-g^(b) 120.0 ± 2.7  0.52 ± 0.03 230.8 ± 14.3 10 1h 6.5N62C-S-Et-β-Glc(Ac)₂ ^(c) 87.7 ± 4.2 1.63 ± 0.15 53.8 ± 5.8 11 1h 5.5N62C-S-Et-β-Glc(Ac)₃ ^(c) 100.3 ± 3.5  1.86 ± 0.12 53.9 ± 4.0 12 1i 5.5N62C-S-i^(b) 123.0 ± 1.6  1.05 ± 0.03 117.1 ± 3.7  13 1j 5.5N62C-S-Et-β-Gal(Ac)₃ ^(c) 103.4 ± 4.3  2.36 ± 0.17 43.8 ± 3.6 14 1k 5.5N62C-S-k^(b) 64.9 ± 1.5 0.88 ± 0.05 73.8 ± 4.5 15 L217C S₁ — — — 41 ± 10.80 ± 0.04 51 ± 3 16 1a 9.5 L217C-S-β-Glc^(b) 27.7 ± 0.4 0.79 ± 0.0335.1 ± 1.4 17 1a 7.5 L217C-S-β-Glc(Ac)₂ ^(c) 44.9 ± 2.0 0.44 ± 0.06102.0 ± 14.6 18 1a 5.5 L217C-S-β-Glc(Ac)₃ ^(c) 36.3 ± 0.8 0.36 ± 0.03100.8 ± 8.7  19 1b 6.5 L217C-S-b^(b) 57.8 ± 0.6 0.67 ± 0.02 86.3 ± 2.720 1c 6.5 L217C-S-c^(b) 50.6 ± 0.9 0.67 ± 0.03 75.5 ± 3.6 21 1d 6.5L217C-S-d^(b) 62.0 ± 1.3 0.55 ± 0.03 112.7 ± 6.6  22 1e 6.5L217C-S-e^(b) 46.2 ± 0.8 0.63 ± 0.08 73.3 ± 3.7 23 1f 6.5 L217C-S-f^(b)30.4 ± 0.6 0.46 ± 0.03 66.1 ± 4.5 24 1g 5.5 L217C-S-Et-α-Glc(Ac)₂ ^(c)72.7 ± 3.1 0.73 ± 0.08  99.6 ± 11.8 25 1h 5.5 L217C-S-Et-β-Glc(Ac)₃ ^(c)29.4 ± 0.8 0.93 ± 0.06 31.6 ± 2.2 26 1i 5.5 L217C-S-Et-α-Man(Ac)₃ ^(c)97.8 ± 2.4 0.59 ± 0.04 165.8 ± 12.0 27 1j 5.5 L217C-S-Et-β-Gal(Ac)₃ ^(c)39.2 ± 0.8 1.17 ± 0.05 33.5 ± 1.6 28 1k 5.5 L217C-S-Et-Lac(Ac)₆ ^(c)27.1 ± 0.6 0.69 ± 0.04 39.3 ± 2.4 29 S156C S₁ — — — 125 ± 4  0.85 ± 0.06147 ± 11 30 1a 9.5 S156C-S-a^(b) 54.8 ± 1.3 0.70 ± 0.04 78.3 ± 4.8 31 1b6.5 S156C-S-b^(b) 77.0 ± 1.2 0.84 ± 0.03 91.7 ± 3.6 32 1c 6.5S156C-S-c^(b) 76.6 ± 1.7 0.73 ± 0.04 104.9 ± 6.2  33 1d 6.5S156C-S-d^(b) 88.6 ± 2.8 0.79 ± 0.06 112.2 ± 9.2  34 1e 6.5S156C-S-e^(b) 78.9 ± 1.9 0.89 ± 0.04 89.7 ± 4.4 35 1f 6.5 S156C-S-f^(b)63.6 ± 1.4 0.89 ± 0.05 71.8 ± 4.3 36 1g 5.5 S156C-S-g^(b) 43.6 ± 0.80.78 ± 0.04 55.9 ± 3.0 37 1h 5.5 S156C-S-h^(b) 64.0 ± 1.3 0.72 ± 0.0488.9 ± 5.2 38 1i 5.5 S156C-S-i^(b) 60.3 ± 0.9 0.71 ± 0.03 84.9 ± 3.8 391j 5.5 S156C-S-j^(b) 51.9 ± 0.6 0.61 ± 0.02 85.1 ± 3.0 40 1k 5.5S156C-S-k^(b) 53.6 ± 0.8 0.79 ± 0.03 67.4 ± 2.8 41 S166C S₁ — — — 42 ± 10.50 ± 0.05 84 ± 9 42 1a 9.5 S166C-S-a^(b) 33.8 ± 1.3 0.66 ± 0.06 51.2 ±5.0 43 1b 6.5 S166C-S-b^(b) 81.9 ± 1.1 1.14 ± 0.03 71.8 ± 2.1 44 1c 6.5S166C-S-c^(b) 67.0 ± 2.2 0.99 ± 0.07 67.6 ± 5.3 45 1d 6.5 S166C-S-d^(b)76.5 ± 2.0 1.17 ± 0.07 65.4 ± 4.3 46 1e 6.5 S166C-S-e^(b) 62.2 ± 1.41.08 ± 0.05 57.6 ± 3.0 47 1f 6.5 S166C-S-f^(b) 58.2 ± 1.2 1.02 ± 0.0457.1 ± 2.5 48 1g 5.5 S166C-S-Et-α-Glc(Ac)₃ ^(c) 31.0 ± 0.8 0.77 ± 0.0540.3 ± 2.8 49 1h 6.5 S166C-S-Et-β-Glc(Ac)₂ ^(c) 95.0 ± 2.1 0.87 ± 0.05109.2 ± 6.7  S166C-S-Et-β-Glc(Ac)₃ ^(d) 50 1h 5.5 S166C-S-Et-β-Glc(Ac)₂^(c) 72.9 ± 1.7 0.65 ± 0.04 112.2 ± 7.4  S166C-S-h^(c) 51 1i 5.5S166C-S-Et-α-Man(Ac)₃ ^(c) 67.7 ± 1.9 1.64 ± 0.09 41.3 ± 2.5 52 1j 5.5S166C-S-Et-β-Gal(Ac)₃ ^(d) 65.1 ± 0.9 0.80 ± 0.03 81.3 ± 3.3 53 1k 5.5S166C-S-Et-Lac(Ac)₅ ^(d) 67.4 ± 1.6 1.65 ± 0.07 40.8 ± 2.0

[0115] The CMMs each appeared as a single band on non-denaturinggradient PAGE, thereby establishing their high purities. The activeenzyme concentration of the resulting CMM solutions was determined byactive site titration with α-toluenesulfonyl fluoride (PMSF) using afluoride ion-sensitive electrode (Hsia et al. “Active-Site Titration ofSerine Proteases Using a Fluoride-Ion Selective Electrode and SulfonylFluoride Inhibitors,” Anal. Biochem., 242:221-227 (1996), which ishereby incorporated by reference). In all cases, modification with thefully deprotected reagents 1b-f (See FIG. 8) led to site-specificglycosylations and the formation of single glycoforms. Furthermore,modification with the protected MTS reagents 1a, g-k (See FIG. 8) gaveproducts with controllable levels of acetylation. Through adjustment ofpH and appropriate selection of the glycosylation site, differentlyacetylated glycoforms of SBL were prepared. This ability to modulate thelevel of acetylation through pH-control vastly expands the structuralvariety of glyco-CMMs that can be conveniently accessed and its scopewas probed through the reaction of 1a (See FIG. 8) with SBL-N62C,-S156C, -S166C, -L217C (FIG. 9).

[0116] The extent of deacetylation during modification was highlysite-dependent.

[0117] Modification of L217C with reagent 1a (See FIG. 8) at pH 9.5 wasaccompanied by complete in situ deacetylation, and the sole product wasthe fully deprotected glucosylated-SBL, L217C-S-β-Glc. In contrast,treatment of N62C, S156C, and S166C with 1a (See FIG. 8) at pH 9.5yielded only fully acetylated products, N62C-S-a (See FIG. 8), S156C-S-a(See FIG. 8), and S166C-S-a (See FIG. 8), respectively. To examine theeffects of pH upon deacetylation, the reaction of L217C with 1a (SeeFIG. 8) was chosen. At pH 7.5 and 5.5, the products retained two andthree acetate groups, forming L217C-S-β-Glc(Ac)2 and L217C-S-β-Glc(Ac)₃,respectively. In all cases, complete integrity of the site selectivitywas retained.

[0118] This valuable site-dependent deacetylation was attributed to anovel intramolecular SBL-catalyzed process. Although acetate esters aremoderately chemically labile in aqueous solution at pH 9.5, they are notat either pH 7.5 or 5.5 (Greene et al., Protective Groups in OrganicSynthesis, 2nd ed. New York, Wiley (1991), which is hereby incorporatedby reference). The striking differences in behavior during modificationbetween L217C and the three other mutants N62C, S1556C, and S166C underidentical reaction conditions discounted the possibility ofdeacetylation prior to modification. In addition, it was noted thatposition 217 bears an internally-oriented side chain and thatmodification of surface exposed position 156 showed no sign ofdeacetylation. This observation discounted both the possibility ofeither in situ chemical deacetylation or intermolecular enzymaticdeacetylation. Furthermore, this ability of SBL to intramolecularlydeacetylate was confirmed by the reaction of L217C-S-β-Glc(Ac)₃ at pH9.5. Incubation of L217C-S-β-Glc(Ac)₃ under standard modificationreaction conditions, but without reagent 1a (See FIG. 8), gaveL217C-S-β-Glc as the sole product (FIG. 9).

[0119] The enormous potential of this method was demonstrated by thepreparation of a small library of differently acetylated glycosylatedCMMs through the reaction of SBL-N62C, -S156C, -S166C, and -L217C withMTS reagents 1g-k (See FIG. 8). Using the pH-activity profiles ofwild-type (“WT”) and CMMs of SBL as a guide, pH 5.5 and 6.5 were chosento minimize deacetylation. Typically, the specific activity of SBL andits CMMs drops sharply below pH 7.5 to levels that at pH 5.5 are 5-20%those at optimal pH (8.5-9.5) (Desantis et al., “Chemical Modificationsat a Single Site Can Induce Significant Shifts in the pH Profiles of aSerine Protease,” J. Am. Chem. Soc., 120:8582-8586 (1998), which ishereby incorporated by reference). As expected, this drop in hydrolyticactivity was reflected in the products of these modifications with 1g-k(See FIG. 8), which in all cases retained two or more acetate groups.

[0120] For example, at pH 5.5 reactions of SBL-L217C and -S166C createdsingly deacetylated CMMs, with the exception of pure dideacetylatedglyco-CMMs L217C-S-Et-α-Glc(Ac)₂, and S166C-S-Lac(Ac)₅. The formation ofthe latter may reflect the presence of two primary acetates indisaccharidic MTS reagent 1k (See FIG. 8). Primary acetate groups aretypically more labile than secondary acetate groups under conditions ofintermolecular enzymatic deacetylation (Bashir et al., “EnzymaticEsterification and De-Esterification of Carbohydrates—Synthesis of aNaturally-Occurring Rhamnopyranoside of P-Hydroxybenzaldehyde and aSystematic Investigation of Lipase-Catalyzed Acylation of SelectedArylpyranosides,” J. Chem. Soc. Perkin-Trans. I, 2203-2222 (1995), whichis hereby incorporated by reference). In contrast, reactions ofSBL-S156C gave only the fully acetylated CMMs, S156C-S-g-k (See FIG. 8).This uniform lack of deacetylation observed for surface exposed glycansat position 156 was consistent with an intramolecular enzyme-catalyzedmechanism requiring internally-oriented acetate groups. Interestingly,the reactions of SBL-N62C were also determined by the anomericconfiguration of 1g-k (See FIG. 8) with α-MTS reagents 1g,i (See FIG. 8)giving products that retained all acetate groups while β-MTS reagents1h,j,k (See FIG. 8) were monodeacetylated. The range of accessibleacetylated glyco-CMMs was further extended through modification at pH6.5. For example, at position 62, it allowed the introduction ofdiacetylated β-glucose, forming N62C-S-Et-β-Glc(Ac)₂, in place of thetriacetylated N62C-S-Et-β-Glc(Ac)₃ formed at pH 5.5. The range ofacetylation at position 166 was similarly expanded through the formationS166C-S-Et-β-Glc(Ac)₂ in place of S166C-S-Et-P-Glc(Ac)₃.

Example 10 Glycan Structure-Hydrolytic Activity Relationships

[0121] The effects of glycosylation upon SBL were assessed by thedetermination of k_(cat) and K_(M) for the hydrolysis ofsuccinyl-AAPF-p-nitroanilide (Suc-AAPF-pNA) at pH 8.6. The kineticparameters of the 48 CMMs generated were compared with those of WT andunmodified mutants in Table 3. The excellently selective and controlledmethod shown in FIG. 8 allowed the introduction of structurally relatedmonosaccharides, D-glucose, D-galactose, and D-mannose, in addition tothe more sterically bulky disaccharide lactose. From the resultingglycosylated CMMs, a detailed and precise set of structure-activityrelationships was generated (FIGS. 10-12).

[0122] At position 62, in the S₂ pocket, the 2.3-fold reduction ink_(cat)/K_(M) caused by mutation to cysteine was partially restored byglycosylation (FIG. 10A). The introduction of ethyl-tethered α- orβ-glucose, β-galactose or α-mannose to N62C increased k_(cat)/K_(M) andformed N62C-S-b-e (See FIG. 8) with k_(cat)/K_(M)s 1.5- to 2-fold lowerthan WT. Despite its steric bulk and high hydrophilicity, disaccharidiclacto-CMM N62C-S-f (See FIG. 8) also showed higher activity than N62Cwith k_(cat)/K_(M) only 1.7-fold lower than WT.

[0123] The effects of the mutation of position 217 in the S₁′ pocketwere intrinsically more dramatic as indicated by a value of k_(cat)K_(M)for L217C that is 4-fold lower than WT (FIG. 10B). The introduction ofdeprotected untethered glucose, forming L217C-S-β-Glc, loweredk_(cat)/K_(M) further to 6-fold lower than WT. In contrast,glycosylation of position 217 with ethyl tethered MTS reagents 1b-f (SeeFIG. 8) restored activity and k_(cat)/K_(M)s for L217C-S-b-f (See FIG.8) were similar to each other in the range 2.5- to 3.1-fold lower thanWT. This striking difference between tethered L217C-S-b-f (See FIG. 8)and untethered L217C-S-β-Glc illustrated that SBL tolerates thereplacement of hydrophobic Leu with highly hydrophilic carbohydratemoieties when they are linked by a hydrophobic ethyl spacer group betterthan directly-linked Cys-S-β-Glc. This may indicate that a structuralrequirement for efficient amidase activity is a closely-boundhydrophobic residue in the S₁′ subsite of SBL and contrasts sharply withthe excellent enhancement of esterase activity caused by the sameCys-S-β-Glc substitution.

[0124] Mutation of position 156 in the S₁ pocket to cysteine caused a1.4-fold drop in k_(cat)/K_(M) (FIG. 10C). Subsequent introduction ofdeprotected S-Et-α-Glc, side chain b (See FIG. 8), resulted in ak_(cat)/K_(M) for S156C-S-b (See FIG. 8) that was 2.3-fold lower thanWT. From S156C-S-b to -f (See FIG. 8), k_(cat)/K_(M)s varied in an arcedmanner peaking at 1.9-fold lower than WT for S156C-S-d (See FIG. 8) andthen decreasing monotonically to a k_(cat)/K_(M) for S156C-S-f (See FIG.8) that was 3-fold lower than WT. The similar K_(M) values for theseS156 CMMs to those of SBL-WT were indicative of these modificationshaving little effect upon ground state binding and were consistent withthe surface exposed orientation of the S156 side chain.

[0125] At position 166, in the S₁pocket, the 2.5-fold decrease ink_(cat)/K_(M) caused by mutation to cysteine was amplified bymodification with 1b (See FIG. 8) and led to a k_(cat)/K_(M) value3-fold lower than WT for S166C-S-b (See FIGS. 8 and 10D). From S166C-S-bto -f (See FIG. 8), k_(cat)/K_(M) decreased monotonically to ak_(cat)/K_(M) for S166C-S-f (See FIG. 8), in which the S. binding sitewas occupied by the sterically bulky disaccharide lactose, that was3.8-fold lower than WT.

Example 11 Kinetic Effects of Glycosylation with AcetylatedCarbohydrates

[0126] The enormous potential of the controlled site-selectiveglycosylation approach depicted in FIG. 8 was illustrated by the greatvariety of changes in k_(cat)/K_(M) that were caused by the introductionof acetylated side chains a,g-k (See FIG. 8) to SBL. These dramaticchanges contrast with the slight variations found for deprotected sidechains b-f (See FIG. 8). For example, at position 62, an alternatingdecrease-increase pattern was observed (FIG. 11A). This resulted in ak_(cat)/K_(M) for tetraacetylated α-gluco-CMM N62C-S-g (See FIG. 8) thatwas 1.1-fold higher than WT. Similar alternating patterns were also seenat positions 217 (FIG. 11B) and 166 (FIG. 11D). At position 156,variations were slight, which was consistent with its surface exposedorientation (FIG. 11C).

[0127] To examine the cause of these variations, the k_(cat)/K_(M)s ofacetylated glycosylated CMMs were compared with those for deprotectedglycosylated CMMs with the same glycan structure and stereochemistry(FIG. 12). This separated the effects of acetylation from the effects ofglycosylation and allowed the underlying effects of modification to bedissected. It was clear from FIG. 12 that the anomeric stereochemistryof the acetylated glycans modulates k_(cat)/K_(M).

[0128] For example, at position 62 (FIG. 12A) comparison of N62C-S-b,c(See FIG. 8) with N62C-S-g (See FIG. 8) and N62C-S-Et-β-Glc(Ac)_(2.3)showed that increasing the number of acetate groups from zero to four,from N62C-S-b (See FIG. 8) to N62C-S-d (See FIG. 8), increasedk_(cat)/K_(M) 1.6-fold for the c-gluco side-chain b (See FIG. 8). Incontrast, increasing the number from zero to two or three, from N62C-S-cto N62C-S-Et-β-Glc(Ac)_(2 or 3), was detrimental for the β-glucoside-chain c (See FIG. 8) and led to a 2-fold decrease. Similarly,N62C-S-Et-β-Gal(Ac)₃ displayed a distinctly lower k_(cat)/K_(M) thanN62C-S-e (See FIG. 8) that was 5-fold lower than WT. These changes ink_(cat)/K_(M) upon acetylation were manifested largely through increasedor decreased ground state binding, of which the most striking examplewas a K_(M) for N62C-S-Et-β-Gal(Ac)₃ that was 2.4-fold higher thandeprotected galacto-CMM N62C-S-e (See FIG. 8).

[0129] At position 217, control of the level of acetylation through pH,as shown in FIG. 9, had allowed the introduction at position 217 ofuntethered β-D-glucose bearing zero, two, and three acetate groups. AsFIG. 12B illustrates, the addition of two or three acetate groupsrestored k_(cat)/K_(M) from 6-fold lower than WT for L217C-S-β-Glc to2-fold lower than WT for L217C-S-/-Glc(Ac)₂ or L217C-S-β-Glc(Ac)₃. Thisshowed that acetylation allowed fine-tuning of activity and paralleledincreases in the esterase k_(cat)/K_(M)s of these CMMs.

[0130] The same trend in k_(cat)K_(M) was observed for the L217C ethyltethered CMMs as at position 62: acetylation was beneficial toa-tethered but detrimental to β-tethered CMMs. For example, increasingthe number of acetate groups in the a-linked glucose moiety from zero totwo, i.e., from L217C-S-b (See FIG. 8) to L217C-S-Et-α-Glc(Ac)₂,increased k_(cat)/K_(M) to 2-fold lower than WT. In contrast, increasingthe number of acetate groups in the epimeric β-linked moiety from zeroto three, i.e., from L217C-S-c (See FIG. 8) to L217C-S-Et-β-Glc(Ac)₃,halved k_(cat)K_(M) to 6-fold lower than WT. Similarly, thek_(cat)/K_(M) of α-linked L217C-S-Et-α-Man(Ac)₃ was 1.5-fold higher thanthe corresponding deprotected L217C-S-d (See FIG. 8), while β-linkedL217C-S-Et-β-Gal(Ac)₃ was 2-fold lower than the correspondingdeprotected L217C-S-e (See FIG. 8).

[0131] Consistent with its surface exposed orientation, the changes atposition 156 caused by acetylation were slight and no variation withanomeric stereochemistry was seen (FIG. 12C). Increasing the number ofacetate groups from zero to four, from S156C-S-b-e (See FIG. 8) toS156C-S-g-j (See FIG. 8), decreased k_(cat)/K_(M) by 1.05- to 1.6-fold.The most sterically bulky lactose side chain (-k) (See FIG. 8) gave riseto the lowest k_(cat)/K_(M) at position 156, 3.2-fold lower than WT, andindicated that even at the surface of SBL the introduction of stericallybulky groups still allowed tailoring of k_(cat)/K_(M).

[0132] At position 166, the effects of increased acetylation were, as atpositions 62 and 217, modulated by glycan anomeric configuration (FIG.12D). However, the direction of these increases and decreases wasreversed: acetylation was beneficial to β-tethered but detrimental toa-tethered CMMs. For example, the a-tethered S166C-S-Et-α-Glc(Ac)₃ had a1.8-fold lower k_(cat)/K_(M) value than the corresponding deprotectedS166C-S-b (See FIG. 8), while β-linked CMMs S166-S-Et-β-Glc(Ac)_(2.3)had 1.6-fold higher k_(cat/)K_(M) values than the corresponding fullydeprotected S166-S-c (See FIG. 8). Again, these variations were largelymanifested through changes in ground state binding. For example, K_(M)increased 1.4-fold from S166C-S-Et-α-Man (-d) (See FIG. 8) toS166C-S-Et-α-Man(Ac)₃.

[0133] It should be noted that the changes in activity of the lacto-CMMsupon acetylation fell largely outside of these trends and at all fourpositions acetylation of the bulky, disaccharidic side-chain f (See FIG.8) caused a general decrease in k_(cat)/K_(M)s. For example, at position62 heptaacetylation, from N62C-S-f (See FIG. 8) to N62C-S-k (See FIG.8), resulted in a lowering of k_(cat)/K_(M) to a value that is 3-foldlower than WT. Despite the greater steric bulk of side chain k (See FIG.8), this drop was a consequence of a lower keg 2-fold lower than N62C-f(See FIG. 8), rather than a higher K_(M). In fact, the K_(M) value ofN62C-S-k (See FIG. 8) was 1.3-fold lower than N62C-S-f (See FIG. 8).Similarly, L217C-S-Et-Lac(Ac)₆ and S166C-S-Et-Lac(Ac)₅ had 1.7-fold and1.4-fold lower k_(cat)/K_(M)s than the corresponding deprotected CMMs,respectively.

[0134] In summary, the strategy of site-directed mutagenesis combinedwith chemical modification was exploited for the site-selectiveglycosylation of SBL. This method was general, versatile and allowed thepreparation of pure glycoforms which constitute the first examples ofregio- and glycan- specific protein glycosylation at predeterminedsites. Careful control of a novel SBL-catalyzed intramoleculardeacetylation greatly expanded the scope of this method and throughreaction of SBL-N62C, -S156C, -S166C, and -L217C with peracetylated MTSreagents 1a,g-k (See FIG. 8) allowed the introduction of glycans withprecisely modulated levels of acetylation.

[0135] The glycosylated CMMs formed display k_(cat)/K_(M) values thatranged from 1.1-fold higher than WT to 7-fold lower than WT. Without theuse of this highly selective glycosylation technique, the determinationof such precise trends would be unachievable and variations caused byprevious non-specific glycosylation could only be interpreted in ageneral manner. It has been demonstrated that subtle differences incarbohydrate structure may be used to fine tune the activity of SBL. Forexample, the anomeric stereochemistry of the glycans introducedmodulated changes in k_(cat)/K_(M) upon acetylation. At positions 62 and217, acetylation enhanced the activity of α-tethered CMMs but decreasedthat of β-tethered. This trend was reversed at position 166 where, incontrast, acetylation enhanced the k_(cat)/K_(M)s of β-tethered CMMs butdecreased those of α-tethered. Consistent with its surface exposednature, changes at position 156 were more modest, but still allowedcontrol of activity particularly through glycosylation with disaccharidelactose. These results illustrated the great potential for tailoringactivity through the correct choice of glycan and glycosylation site.

[0136] The ability of the glycosylation method to glycosylate thebinding pockets of SBL also creates opportunities to broaden itssubstrate specificity. For instance, an array of hydrogen bondinghydroxyl groups may broaden its specificity towards hydrogen bondingsubstrates such as glycosylated amino acids. Subtilisins have beenelegantly used to catalyze the synthesis of glycopeptides (Witte et al.,“Solution- and Solid-Phase Synthesis of N-protected Glycopeptide Estersof the Benzyl Type as Substrates for Subtilisin-Catalyzed GlycopeptideCouplings,” J. Am. Chem. Soc. 120:1979-1989 (1998); Wong et al.,“Enzymatic-Synthesis of N-Linked and O-Linked Glycopeptides,” J. Am.Chem. Soc. 115:5893-5901 (1993), which are hereby incorporated byreference). However, the natural specificity of these enzymes haslimited these peptide ligations to those in which the glycosylatedresidues are at least one residue distant (P₂, P₃ . . . or P₂′, P₃′ . .. ) from the amide bond formed. For example, while ligation of Z-Gly-OBzwith H-Gly-Ser(Ac₃GlcNAcβ)-NH, was successful, no yield of product wasobtained with H-Ser(Ac₃GlcNAcp3)-NH₂ (Witte et al., J. Am. Chem. Soc.,120:1979-1989 (1998), which is hereby incorporated by reference). Theintroduction of sugars to the S₁ and S₁′ subsites as hydrogen bondinggroups demonstrated here may enhance the specificity of proteasestowards hydrophilic substrates.

[0137] Furthermore, by choosing carbohydrate attachments that differfrom each other at only one stereocenter, SARs may be determined byexamining changes in activity as the nature of sugar side chain isvaried. For example, the effect of inverting stereocenters in the orderC-4→C-1→C-2 can be determined using CMMs in the series e→c→b→d (See FIG.8). While the current illustrations have been with SBL as a proteinexample, the method is clearly amenable to the glycosylation of anyprotein and is without limitation with respect to the sites and to theglycans that may be conjugated. It will, therefore, allow theintroduction of any therapeutically important carbohydrate recognitiondeterminant, of which the P-D-galactopyranosyl moiety of e and f (SeeFIG. 8) that represents a ligand of the hepatic asialoglycoproteinreceptor (Sharon et al., Essays Biochem., 30:59-75 (1995), which ishereby incorporated by reference) is just one example.

Example 12 Esterase Screen

[0138] Specificity constants determined using the low substrateapproximation were measured indirectly using Ellman's reagent (Ellman etal., Biochem. Pharmacol., 7:88-95 (1961), which is hereby incorporatedby reference) (ε₄₁₂=13600 M⁻¹cm⁻¹) using 0.15 and 0.30 mMsuccinyl-AAPF-SBn as substrate in 0.1 M Tris.HCl, containing 0.005 vol %DMSO, 1 vol % 37.5 mM Ellman's reagent in DMSO, pH 8.6.

Example 13 Full Esterase Kinetics Measurements

[0139] Michaelis-Menten constants were measured at 25° C. by curvefitting (Grafit® 3.03, Erithacus Software Ltd., Staines, Middlesex, UK)of the initial rate data determined at eight concentrations (31.25 μM-3mM) of the succinyl-AAPF-SBn substrate, followed indirectly usingEllman's reagent in 0.1 M Tris.HCl, containing 0.005 vol % DMSO, 1 vol %37.5 mM Ellman's reagent in DMSO, pH 8.6.

Example 14 Esterase Activity Screen

[0140] The glyco-CMMs shown in Table 4 were prepared, by reactingreagents la-k (See FIG. 8) with the chosen cysteine mutants SBL-N62C,-S156C, -S166C and -L217C, purified and extensively characterized asdescribed previously. TABLE 4 Esterase Screen Results for GlycosylatedCMMs^(a) N62C-R L217C-R S166C-R S156C-R k_(cal)/K_(M) k_(cal)/K_(M)k_(cal)/K_(M) k_(cal)/K_(M) -R (s⁻¹mM⁻¹) E/A^(b) (s⁻¹mM⁻¹) E/A^(b)(s⁻¹mM⁻¹) E/A^(b) (s⁻¹mM⁻¹) E/A^(b) H 4380 48 5540 109  350  4 — —-SβGlc — — 6350 186 — — — — B 5327 37 7773  90 2688 37 1984 22 C 6625 629791 130 2732 40 2197 21 D 4627 36 8651  77 2924 45 2648 24 E 5729 4811923  163 3241 56 2505 28 F 5990 48 9080 137 2511 44 1692 24-SβGlc(Ac)₂ — — 8776  86 — — — — -SβGlc(Ac)₃ — — 11642  115 — — — —-SβGlc(Ac)₄ 2502 19 — — 2537 50 1356 17 (-a) -SetαGlc(Ac)₂ — — 7893  79— — — — -SetαGlc(Ac)₃ — — — — 1829 45 — — -SetαGlc(Ac)₄ 6851 30 — — — —1887 34 (-g) -SetβGlc(Ac)₂ 1925 36 — — 5058 46 — — -SetβGlc(Ac)₃ 2736 514935 156 6033 54 — — -SetβGlc(Ac)₄ — — — — — — 2321 26 (-h)-SetαMan(Ac)₃ — — 11751   71 1537 37 — — -SetαMan(Ac)₄ 5807 50 — — — —2629 31 (-i) -SetβGal(Ac)₃ 2169 50 3889 116 3159 38 — — -SetβGal(Ac)₄ —— — — — — 2010 24 (-j) -SetLac(Ac)₃ — — — — 1543 38 — — -SetLac(Ac)₆ — —5468 139 — — — — -SetLac(Ac)₇ 2135 29 — — — — 1516 22 (-k)

[0141] The kinetic parameters of esterase activity were determined at pH8.6 by indirectly following the release of thiobenzyl alcohol from thesubstrate succinyl-Ala-Ala-Pro-Phe-SBn (suc-AAPF-SBn) with Ellman'sreagent (Ellman et al. Biochem. Pharmacol., 7:88-95 (1961), which ishereby incorporated by reference). To allow a rapid screen of esteraseactivity, a low substrate concentration ([S]<<K_(M)) was used thatallowed k_(cat)/K_(M) to be determined directly from the initial rate ofreaction. The results from the screen are shown in Table 4.

[0142] Modification at position 62, in the S₂ pocket, with deprotectedsugar reagents 1b-f (See FIG. 8) increased k_(cat)/K_(M), resulting in aseries of five enzymes that had similar k_(cat)/K_(M)s that were 1.3- to1.9-fold greater than WT (FIG. 13A). The presence of an α-linkage wasclearly deleterious to activity, as N62C-SEtβGtc (-c) (See FIG. 8) had ak_(cat)/K_(M) 1.2-fold greater than its epimer N62C-SEtαGlc (-b) (SeeFIG. 8) and 1.9-fold greater than WT. Furthermore, the a-linkedN62C-SEtαMan (-d) (See FIG. 8) had the lowest k_(cat)/K_(M) in thisgroup which was 1.3-fold greater than WT.

[0143] As at position 62, the introduction of any of the deprotectedsugar side chains b-f (See FIG. 8) at position 217, in the S₁′ pocketincreased k_(cat)K_(M) (FIG. 13B). However, the effects of glycosylationat this site were far more dramatic as demonstrated by a k_(cat)/K_(M)for L217C-SEtβGal (-e) (See FIG. 8) that was 3.4-fold greater than WT.By comparing the k_(cat)/K_(M)s of L217C-SβGlc and L217C-SEtβGlc (-c)(See FIG. 8), it was possible to gauge the effect on activity ofintroducing an ethyl tether at this position. This introductionincreased k_(cat)/K_(M), from 1.8-fold greater than WT for L217C-SβGlcto 2.7-fold greater than WT for L217C-SEtβGlc (-c) (See FIG. 8). As atposition 62, β-linked glyco-CMMs (-c,-e,-f) (See FIG. 8) had higherk_(cat)/K_(M)s than the α-linked ones (-a,-d) (See FIG. 8). For example,L217C-SEtβGlc (-c) (See FIG. 8) had a k_(cat)/K_(M) 1.3-fold greaterthan L217C-SEtαGlc (-b) (See FIG. 8).

[0144] Consistent with the surface exposed orientation of the S156 sidechain, the S156C deprotected glyco-CMMs had similar k_(cat)/K_(M)s thatwere 1.3- to 2.1-fold lower than WT (FIG. 13C).

[0145] At position 166, in the S₁pocket, mutation to cysteine resultedin an enzyme with a severely lowered k_(cat)/K_(M) that was 10-foldlower than WT. However, subsequent modification with 1b-f (See FIG. 8)restored much of the catalytic activity (FIG. 13D), and the S166Cdeprotected glyco-CMMs S166C-S-b-f (See FIG. 8) had similark_(cat)/K_(M)s, that varied from 1.1- to 1.4-fold lower than WT.

Example 15 Kinetic Effects of Glycosylation with AcetylatedCarbohydrates

[0146] At position 62, in the S₁pocket, in sharp contrast to the trendobserved for the unacetylated N62C CMMs, acetylated N62C CMMs had a widerange of k_(cat)/K_(M)s. Introduction of acetates increased thek_(cat)/K_(M)s of the α-linked CMMs relative to the correspondingdeprotected glyco-CMMs (FIG. 14A). Thus, N62C-SEtαGlc(Ac)₄ (-b) (SeeFIG. 8) and N62C-SEtαMan(Ac)₄ (-d) (See FIG. 8) had k_(cat)/K_(M)s 1.9-and 1.6-fold greater than WT, respectively. Acetylation was clearlydeleterious for β-linked CMMs, as N62C-SEtβGal(Ac)₃, N62C-SEtβGlc(Ac)₂,and N62C-SEtβGlc(Ac)₃ all had k_(cat)/K_(M)s lower than WT. However,increasing the number of acetates present on the CMM restored activity:N62C-SEtβGlc(Ac)₃ had a k_(cat)/K_(M) only 1.3-fold lower than WT and1.5-fold higher than N62C-SEtβGlc(Ac)₂. In spite of their size, thesterically bulky side chain lactosylated N62C CMMs, N62C-SEtLac (-f)(See FIG. 8) and N62C-SEtLac(Ac)₇ (-k) (See FIG. 8) had k_(cat)/K_(M)sthat were similar to those of the CMMs derived from monosaccharides.This provided a clear example of the versatility of the glycosylationmethod illustrated in FIG. 8 and demonstrated that by using this methodit was possible to introduce very large structures into the active siteof SBL while maintaining catalytic competency.

[0147] Modification with acetylated reagents 1a,g-k (See FIG. 8) atposition 217, in the S₁′ pocket, as with 1b-k (See FIG. 8), led to CMMswith greater than WT k_(cat)K_(M)s (FIG. 14B). For the untetheredglyco-CMMs, increasing the number of acetates dramatically increasedk_(cat)/K_(M), from 1.8-fold greater than WT for L217C-SβGlc to 2.4-foldgreater than WT for L217C-SβGlc(Ac)₂ and to 3.2-fold greater than WT forL217C-SβGlc(Ac)₃ and mirrored the trend seen in amidase kinetics. Forthe ethyl linked L217C glyco-CMMs, the effect of acetylation wasdependent on the anomeric stereochemistry, as observed for N62Cglyco-CMMs and the L217C glyco-CMMs amidase kinetics. Acetylation ofα-linked CMMs increased k_(cat)/K_(M) but decreased k_(cat)/K_(M) forβ-linked CMMs. This was most pronounced for L217C-SEtβGal(Ac)₃ which hada k_(cat)/K_(M) only 1.1-fold greater than WT and 3.1-fold lower thanL217C-SEtβGal (-e). In contrast to the effect on deprotected L217Cglyco-CMMs, the activity of acetylated L217C glyco-CMMs decreased uponthe introduction of the ethyl linker. For example, L217C-SβGlc(Ac)₃ hada k_(cat)/K_(M) 3.2-fold greater than WT, as compared with the 1.4-foldgreater than WT k_(cat)/K_(M) of L217C-SEtβGlc(Ac)₃.

[0148] At position 156, in the S₁ pocket, the S156C acetylatedglyco-CMMs displayed little difference in their kinetic constants fromtheir unacetylated counterparts (FIG. 14C), an observation that wasconsistent with the surface exposed nature of the position 156 sidechain. Introducing the ethyl linker led to an increase in k_(cat)/K_(M)from 2.6-fold lower than WT for S156C-SβGlc(Ac)₄ (-a) (See FIG. 8) to1.5-fold lower than WT for S156C-SEtβGlc(Ac)₄ (-h) (See FIG. 8).

[0149] In general, at position 166, in the S₁ pocket, the effect ofacetylation on 0 o S166C ethyl linked glyco-CMMs was to reducek_(cat)/K_(M) relative to their deprotected counterparts (FIG. 14D). Theexceptions were the ethyl linked β-gluco-CMMs, S166C-SEtβGlc(Ac)₂ andS166C-SEtβGlc(Ac)₃, which displayed k_(cat)/K_(M)s 1.4- and 1.7-foldgreater than WT, respectively. These were the only two glyco-CMMsprepared at this site to show an enhancement in k_(cat)/K_(M) relativeto WT, and this example illustrates that the correct selection of sugaris crucial to the tailoring of enzyme activity. In contrast to theeffects observed at positions 62 and 217, an β-linkage to the sugarmoiety was deleterious to the activity of the acetylated CMMs andS166C-SEtαGlc(Ac)₃ had a k_(cat)/K_(M) 1.9-fold lower than WT, in directcontrast to S166C-SEtβGlc(Ac)₃. Introduction of the sterically bulkylactose moiety, in both acetylated and unacetylated forms, led to CMMsS166C-SEtLac (-f) (See FIG. 8) and S166C-SEtLac(Ac), with lowk_(cat)/K_(M)s that were 1.4- and 2.3-fold lower than WT, respectively.

Example 16 Full Esterase Kinetics

[0150] The three esterases with the highest k_(cat)/K_(M)s determined bythe above screen, L217C-SβGlc(Ac)₃, L217C-SEtαMan(Ac)₃, andL217C-SEtβGal (-e) (See FIG. 8) had their individual k_(cat)s and K_(M)sdetermined by the initial rates method. The results are shown in Table5. TABLE 5 Full Esterase Kinetic Data for Glycosylated CMMs^(a)k_(cal)/K_(M) Enzyme k_(cal) (s⁻¹) K_(M) (mM) (mM⁻¹s⁻¹) × WT E/A^(b) WT1940.0 ± 180   0.54 ± 0.07 3592.5 ± 572.7 1  17 L217C-SβGlc(Ac)₃ 4427.5± 100.9 0.15 ± 0.01 29516.7 ± 2079.0 8.4 293 L217C-SEtαMan(Ac)₃ 3827.0 ±59.5  0.30 ± 0.01 12756.7 ± 469.2  3.6  77 L217C-SetβGal 4398.5 ± 189.80.36 ± 0.04 12218.1 ± 1456.3 3.5 167

[0151] The results were in good agreement with those determined by thescreen for L217C-SEtαMan(Ac)₃ and L217C-SEtβGal (-e) (See FIG. 8) andconfirmed the activity of these two enzymes to be 3.6- and 3.5- foldhigher than WT, respectively. These increases in activity arose fromboth increased transition state stabilization, with k_(cat)s 2- and2.3-fold greater than WT, respectively, and from greater substratebinding, with K_(M)s 1.8- and 1.5-fold lower than WT, respectively.

[0152] Remarkable results were obtained for L217C-SβGlc(Ac)₃. Thisenzyme had a k_(cat) 2.3-fold greater than WT and a K_(M) 3.6-fold lowerthan WT, giving a k_(cat)/K_(M) 8.4-fold greater than WT and some2.5-fold greater than the value estimated by the screen. The differencein parameters obtained from the screen and the full kinetic analysisexposed a limitation of the low substrate screen. For the low substrateapproximation to be accurate, the substrate concentration must be smallcompared to K_(M). The K_(M) of L217C-SβGlc(Ac)₃ (0.15 mM) was evidentlyso small that the approximation did not hold in this case. This was thelargest enhancement of activity relative to WT achieved using thecombined site-directed mutagenesis and chemical modification strategy.

Example 17 Esterase Activity Versus Amidase Activity

[0153] The differing effects of glycosylation upon amidase and esterasek_(cat)/K_(M) can be compared in an informative manner using the(k_(cat)/K_(M))_(esterase)/(k_(cat)K_(M))_(amidase) ratio, E/A (see FIG.15).

[0154] All N62C deprotected glyco-CMMs had E/As that were enhancedrelative to WT. The increase in E/A was dependent on the presence ofeither an α- or a β-linkage with the β-linked CMMs N62C-SEtβGlc (-c)(See FIG. 8), -SEtβGal (-e) (See FIG. 8), -SEtLac (-f) (See FIG. 8)having higher E/As than the β-linked CMMs N62C-SEtαGlc (-b) (See FIG. 8)and -SEtαMan (-d) (See FIG. 8) (FIG. 15A). These increased ratios weredue to both increases in esterase k_(cat)/K_(M)s and reductions inamidase k_(cat)/K_(M)s.

[0155] As observed for the modifications made at position 62,glycosylation at position 217, in the S₁′ pocket, led to enzymes thathad greatly increased E/A ratios relative to WT. Mutation to cysteine atposition 217 increased E/A to 6.4-fold greater than WT. Modificationwith unacetylated β-linked sugars -SβGlc, -SEtβGlc (-c) (See FIG. 8),-SEtβGal (-e) (See FIG. 8), -SEtLac (-f) (See FIG. 8) further increasedE/A (FIG. 15B). In contrast, a-linked glyco-CMMs had lower E/A valuesthan that of the mutant. These changes in E/As were a result of parallelchanges in esterase k_(cat)/K_(M)s that were further amplified byopposing changes in amidase k_(cat)/K_(M)s. Introduction of an ethyllinker reduced the E/A from 10.9-fold greater than WT for L217C-SβGlc to7.6-fold greater than WT for L217C-SEtβGlc (-c) (See FIG. 8).

[0156] At position 156, in the S₁pocket, E/A ratios for unacetylatedglyco-CMMs were all similar to each other in the range 1.2- to l .6-foldgreater than WT (FIG. 15C). The mutant S166C had an exceptionally lowE/A that was 4.2-fold lower than WT (FIG. 15D) and this was largely aresult of its very low esterase k_(cat)/K_(M). Because modification ofS166C restored esterase k_(cat)/K_(M) to levels approaching that of WTand because S166C glyco-CMMs were poor amidases relative to WT, the netresult was a family of CMMs with similar E/A ratios that were allenhanced relative to WT and significantly higher than the cysteinemutant (FIG. 15D).

Example 18 Effects of Glycosylation with Acetylated Carbohydrates on E/A

[0157] At position 62, in the S₂ pocket, with the exception ofN62C-SEtβGal(Ac)₃ and N62C-SEtαMan(Ac)₄ (-i) (See FIG. 8), acetylationof N62C glyco-CMMs led to a reduction in E/A. However, like theirdeprotected counterparts, the acetylated N62C glyco-CMMs all had largerE/As than WT (FIG. 16A). Increasing the level of acetylation increasedthe E/A ratio from 2.1-fold greater than WT for N62C-SEtβGlc(Ac)₂ to3.0-fold greater than WT for N62C-SEtβGlc(Ac)₃. In spite of the generalincreases in E/A observed for the ethyl linked glyco-CMMs, theuntethered CMM N62C-SβGlc(Ac), (-a) (See FIG. 8) had an E/A very similarto WT.

[0158] At position 217, in the S₁′ pocket, L217C acetylated glyco CMMsall had enhanced E/As relative to WT (FIG. 16B). As for theirdeprotected counterparts. β-linked acetylated glyco-CMMs had higher E/Asthan that of the L217C mutant, whereas the E/As of the α-linkedacetylated glyco-CMMs were lower than that of the mutant. In fact,modification at this site produced CMMs with by far the greatestenhancement of this ratio and the E/A for L217C-SβGlc(Ac)₃ was 17.2-foldgreater than WT. In contrast to the L217C deprotected glyco-CMMs,introducing an ethyl linker lowered E/A from 17.2-fold greater than WTfor L217C-SβGlc(Ac)₃ to 9.2-fold greater than WT for L217C-SEtβGlc(Ac)₃.For the untethered L217C acetylated glyco-CMMs, increasing the number ofacetates also increased E/A, from 5.0-fold greater than WT forL217C-SEtβGlc(Ac)₂ to 17.2-fold greater than WT for L217C-SEtβGlc(Ac)₃.

[0159] At position 156, in the S₁pocket, acetylation of a-linked glycoCMMs increased E/As (FIG. 16C). Hence, S156C-SEtβGlc(Ac)₄ (-h) (See FIG.8) had an E/A that was 1.5 fold greater than WT whereasS156C-SEtαGlc(Ac)₄ (-g) (See FIG. 8) had an E/A 2.0-fold greater thanWT. S156C-SEtαMan(Ac)₄ (-i) (See FIG. 8) also had an E/A 1.3-foldgreater than that of its unacetylated counterpart, S156C-SEtαMan (-d)(See FIG. 8).

[0160] At position 166, in the S₁pocket, acetylation caused an increasein E/A for the glucosylated CMMs, irrespective of the anomericstereochemistry (FIG. 16D). Acetylation of all other sugar moieties ledto a reduction in E/A. Increasing the number of acetates increased E/Afrom 2.7-fold greater than WT for S166C-SEtβGlc(Ac)₂ to 3.2-fold greaterthan WT for S166C-SEtβGlc(Ac)₃.

Example 19 Molecular Modeling

[0161] The X-ray structure of subtilisin Bacillus lentus with thepeptide inhibitor AAPF bound (Brookhaven database entry 1JEA) was usedas the starting point for calculations on wild type and CMMs. The enzymesetup was performed with Insight II, version 2.30 (Biosym Technologies,Inc. San Diego, Calif.). To create initial coordinates for theminimization, hydrogens were added at the pH 8.6 used for kineticmeasurements. This protonated all Lys and Arg residues and theN-terminus and deprotonated all Glu and Asp residues and the C-terminalcarboxyl group. The protonated form of His 64 was used in allcalculations. The model system was solvated with a 5 Å layer of watermolecules. The total number of water molecules in the system was 1143.The overall charge of the enzyme-inhibitor complex resulting from thissetup was +4 for the WT enzyme. Energy simulations were performed withthe DISCOVER program, Version 2.9.5 (Biosym Technologies, Inc., SanDiego, Calif.) on a Silicon Graphics Indigo computer, using theconsistent valence force field (CVFF) function. A non-bonded cutoffdistance of 18 Å with a switching distance of 2 æ was employed. Thenon-bonded pair list was updated every 20 cycles and a dielectricconstant of 1 was used in all calculations. The WT enzyme was minimizedin stages, with initially only the water molecules being allowed tomove, followed by water molecules and the amino acid side chains, andthen finally the entire enzyme. The mutated and chemically modifiedenzymes were generated by modifying the relevant amino acid using theBuilder module of Insight. These structures were then minimized in asimilar manner. Initially, the side-chain of the mutated residue and thewater molecules were minimized. Then, all side-chains and the watermolecules were minimized while the backbones of the residues wereconstrained, then all of the atoms were minimized. The AAPF inhibitorwas free to move throughout all stages of the minimization. Each stageof energy minimization was conducted by means of the method of steepestdescents without Morse or cross terms until the derivative of energywith respect to structural perturbation was less than 5.0 kcal/Å; then,the method of conjugate gradients, without Morse or cross terms untilthe derivative of energy with respect to structural perturbation wasless than 1.0 kcal/Å; and, finally, the method of conjugate gradients,with Morse and cross terms until the final derivative of energy withrespect to structural perturbation was less than 0.1 kcal/Å.

[0162] The molecular basis for the vastly improved esterase activitiesobserved was analyzed by molecular modeling of the peptidyl productinhibitor AAPF bound to the SBL-CMM L217C-SβGlc(Ac)₃. While thesubstrate employed for kinetic analysis is succinylated, this moiety wasnot included in molecular modeling since its orientation was notreported in the X-ray structure of SBL, suggesting high mobility in thecrystal.

[0163] As shown in FIGS. 17 and 18, molecular modeling analysis ofL217C-SβGlc(Ac)₃ revealed that the observed k_(cat)/K_(M) changescorrelate with the occupation of the S₁′ pocket of SBL by theglucosylated -SβGlc(Ac)₃ side chain. In the minimized structure (FIG.17), the position of the glucose moiety was fixed by a network ofhydrogen-bonding interactions (shown as white dotted lines) between theoxygen atoms on the C-3, -4 and 6 substituents of glucose and watermolecules in the surrounding external solvent. This extensive solvationdirected the C-2 substituent of glucose internally towards the catalytictriad. In this orientation, the carbonyl oxygen atom of the C-2 acetategroup acts a hydrogen bond (1.89 Å) acceptor and stabilizes watermolecule 127 in close proximity to the carboxy terminus of AAPF, usedhere as a substrate analog for modeling, and is shown in FIG. 17hydrogen-bonding to the O atom of the carboxylic acid.

[0164] These results suggested that firstly the low amidase activity ofL217C-S-Glc(Ac)₃, which was 2-fold lower than WT, was a result of theS₁′ pocket being occupied by the glucose moiety at position 217. Thisprevents efficient binding of the pNA leaving group and, therefore,decreases the rate of acyl-enzyme intermediate formation which is therate-determining step for amidase activity. Secondly, after the pNA hasbeen displaced to form the covalent Acyl-Ser221 intermediate, theglucose moiety stabilizes a crucial, nucleophilic water molecule (Wat127) in close proximity to the carbonyl carbon atom, through ahydrogen-bond to the oxygen of the C-2 acetate of glucose, asillustrated in FIG. 18. This facilitates hydroylsis of the acyl-enzymeintermediate and therefore increases the rate of deacylation, which isthe rate limiting step for esterase activity (Zerner et al., J. Am.Chem. Soc., 86:3674-3679 (1964); Whitaker et al., J. Am. Chem. Soc.,87:2728-2737 (1965); Berezin et al., FEBS Lett., 15:121-124 (1971),which are hereby incorporated by reference).

[0165] Glycosylation of SBL at sites within the active site candramatically enhance its esterase activity. The library of glycosylatedCMMs synthesized using the combined site directed mutagenesis andchemical modification strategy contains 22 CMMs with greater than WTactivity. Glycosylation at positions 62, in the S₂ pocket, and 217, inthe S₁′ pocket, gave the greatest increases in k_(cat)/K_(M). The mostactive CMM L217C-SβGlc(Ac)₃ had a k_(cat)/K_(M) that was 8.4-foldgreater that WT and was the most active esterase synthesized using thisapproach. When surface exposed position 156 was glycosylated, there waslittle alteration in activity, and this demonstrates that theintroduction of sugars at such sites has little effect on the catalyticactivity of SBL.

[0166] In addition to the tailoring of esterase k_(cat)K_(M) values,glycosylation also led to enormous improvements in specificity for esterversus amide hydrolysis, as determined by measurement of the ratio(k_(cat)/K_(M))_(esterase)/(k_(cat)/K_(M))_(amidase), E/A. This ratiohas been increased to 17.2-fold greater than WT for L217C-SβGlc(Ac)₃.Such enzymes are very attractive candidates for use in peptidesynthesis, where a high esterase to amidase ratio is desirable.Furthermore, the CMMs described above have an even greater potential forthis purpose, as the increases in E/As have been achieved by an increasein the catalytic efficiency of these enzymes towards ester substrates,in addition to a reduction in the amidase activity.

[0167] Although the invention has been described in detail for thepurpose of illustration, it is understood that such detail is solely forthat purpose, and variations can be made therein by those skilled in theart without departing from the spirit and scope of the invention whichis defined by the following claims.

What is claimed:
 1. A chemically modified mutant protein, said mutantprotein comprising a cysteine residue substituted for a residue otherthan cysteine in a precursor protein, the substituted cysteine residuebeing subsequently modified by reacting said cysteine residue with aglycosylated thiosulfonate.
 2. A chemically modified mutant proteinaccording to claim 1, wherein the protein is an enzyme.
 3. A chemicallymodified mutant protein according to claim
 2. wherein the enzyme is aprotease.
 4. A chemically modified mutant protein according to claim 3,wherein the protease is a Bacillus lentus subtilisin.
 5. A chemicallymodified mutant protein according to claim 1, wherein said thiosulfonatecomprises an alkylthiosulfonate.
 6. A chemically modified mutant proteinaccording to claim 5, wherein said alkylthiosulfonate comprisesmethanethiosulfonate.
 7. A chemically modified mutant protein accordingto claim 1, wherein the residue other than cysteine is an amino acidselected from the group consisting of asparagine, leucine, and serine.8. A chemically modified mutant protein according to claim
 1. whereinthe residue other than cysteine is in a substrate binding subsite of theprotein.
 9. A chemically modified mutant protein according to claim 1,wherein the glycosylated thiosulfonate comprises a thiol side chaincomprising -S-β-Glc, -S-Et-β-Gal, -S-Et-β-Glc, -S-Et-α-Glc, -S-Et-α-Man,-S-Et-Lac, -S-β-Glc(Ac)₂, -S-β-Glc(Ac)₃, -S-β-Glc(Ac)₄,-S-Et-α-Glc(Ac)₂, -S-Et-α-Glc(Ac)₃, -S-Et-α-Glc(Ac)₄, -S-Et-β-Glc(Ac)₂,-S-Et-β-Glc(Ac)₃, -S-Et-β-Glc(Ac)₄, -S-Et-α-Man(Ac)₃, -S-Et-α-Man(Ac)₄,-S-Et-β-Gal(Ac)₃, -S-Et-β-Gal(Ac)₄, -S-Et-Lac(Ac)₅, -S-Et-Lac(Ac)₆, or-S-Et-Lac(Ac)₇.
 10. A chemically modified mutant protein according toclaim 1 wherein the carbohydrate moiety is a dendrimer moiety.
 11. Amethod of producing a chemically modified mutant protein comprising thesteps of: (a) providing a precursor protein; (b) substituting an aminoacid residue other than cysteine in said precursor protein with acysteine; (c) reacting said substituted cysteine with a glycosylatedthiosulfonate, said glycosylated thiosulfonate comprising a carbohydratemoiety; and (d) obtaining a modified glycosylated protein wherein saidsubstituted cysteine comprises a carbohydrate moiety attached thereto.12. A method according to claim 11, wherein said thiosulfonate comprisesan alkylthiosulfonate.
 13. A method according to claim 12, wherein saidalkylthiosulfonate comprises a methanethiosulfonate.
 14. A methodaccording to claim 11, wherein the protein is an enzyme.
 15. A methodaccording to claim 14, wherein the enzyme is a protease.
 16. A methodaccording to claim 15, wherein the protease is a Bacillus lentussubtilisin.
 17. A method according to claim 11, wherein the amino acidresidue other than cysteine is an amino acid selected from the groupconsisting of asparagine, leucine, and serine.
 18. A method according toclaim 11, wherein the amino acid residue other than cysteine is in asubstrate binding subsite of the protein.
 19. A method according toclaim 11, wherein the glycosylated thiosulfonate comprises a thiol sidechain comprising -S-β-Glc, -S-Et-β-Gal, -S-Et-β-Glc, -S-Et-α-Glc,-S-Et-α-Man, -S-Et-Lac, -S-β-Glc(Ac)₂, -S-β-Glc(Ac)₃, -S-β-Glc(Ac)₄,-S-Et-α-Glc(Ac)₂, -S-Et-α-Glc(Ac)₃, -S-Et-α-Glc(Ac)₄, -S-Et-β-Glc(Ac)₂,-S-Et-β-Glc(Ac)₃, -S-Et-β-Glc(Ac)₄, -S-Et-α-Man(Ac)₃, -S-Et-α-Man(Ac)₄,-S-Et-β-Gal(Ac)₃, -S-Et-β-Gal(Ac)₄, -S-Et-Lac(Ac)₅, -S-Et-Lac(Ac)₆, or-S-Et-Lac(Ac)₇.
 20. A method according to claim 11, wherein thecarbohydrate moiety is a dendrimer moiety.
 21. A glycosylatedthiosulfonate comprising:

wherein R comprises -β-Glc, -Et-β-Gal, -Et-β-Glc, -Et-α-Glc, -Et-α-Man,-Et-Lac, -β-Glc(Ac)₂, -β-Glc(Ac)₃, -β-Glc(Ac)₄, -Et-α-Glc(Ac)₂,-Et-α-Glc(Ac)₃, -Et-α-Glc(Ac)₄, -Et-β-Glc(Ac)₂, -Et-β-Glc(Ac)₃,-Et-β-Glc(Ac)₄, -Et-α-Man(Ac)₃, -Et-α-Man(Ac)₄, -Et-β-Gal(Ac)₃,-Et-β-Gal(Ac)₄, -Et-Lac(Ac)₅, -Et-Lac(Ac)₆, or -Et-Lac(Ac)₇.
 22. Amethod of modifying the functional characteristics of a proteincomprising: providing a protein and reacting the protein with aglycosylated thiosulfonate reagent under conditions effective to producea glycoprotein with altered functional characteristics as compared tothe protein.
 23. A method according to claim 22, wherein the protein isan enzyme.
 24. A method according to claim 23, wherein the enzyme is aprotease.
 25. A method according to claim 24, wherein the protease is aBacillus lentus subtilisin.
 26. A method according to claim 22, whereinthe glycosylated thiosulfonate comprises:

wherein R comprises -β-Glc, -Et-β-Gal, -Et-β-Glc, -Et-α-Glc, -Et-α-Man,-Et-Lac, -β-Glc(Ac)₂, -β-Glc(Ac)₃, -β-Glc(Ac)₄, -Et-α-Glc(Ac)₂,-Et-α-Glc(Ac)₃, -Et-α-Glc(Ac)₄, -Et-β-Glc(Ac)₂, -Et-β-Glc(Ac)₃,-Et-β-Glc(Ac)₄, -Et-α-Man(Ac)₃, -Et-α-Man(Ac)₄, -Et-β-Gal(Ac)₃,-Et-β-Gal(Ac)₄, -Et-Lac(Ac)₅, -Et-Lac(Ac)₆, or -Et-Lac(Ac)₇.
 27. Amethod of determining the structure-function relationships of chemicallymodified mutant proteins comprising: providing first and secondchemically modified mutant proteins according to claim 1, wherein theglycosylation pattern of the second chemically modified mutant proteinis different from the glycosylation pattern of the first chemicallymodified mutant protein; evaluating a functional characteristic of thefirst and second chemically modified mutant proteins; and correlatingthe functional characteristic of the first and second chemicallymodified mutant proteins with the structures of the first and secondchemically modified mutant proteins.
 28. A method according to claim 27,wherein the protein is an enzyme.
 29. A method according to claim 28,wherein the enzyme is a protease.
 30. A method according to claim 29,wherein the protease is a Bacillus lentus subtilisin.
 31. A methodaccording to claim 27, wherein the residue other than cysteine is anamino acid selected from the group consisting of asparagine, leucine,and serine.
 32. A method according to claim 27, wherein the residueother than cysteine is in a substrate binding subsite of the protein.33. A method according to claim 27, wherein the glycosylatedthiosulfonate comprises a thiol side chain comprising -S-β-Glc,-S-Et-β-Gal, -S-Et-β-Glc, -S-Et-α-Glc, -S-Et-α-Man, -S-Et-Lac,-S-β-Glc(Ac)₂, -S-β-Glc(Ac)₃, -S-β-Glc(Ac)₄, -S-Et-α-Glc(Ac)₂,-S-Et-α-Glc(Ac)₃, -S-Et-α-Glc(Ac)₄, -S-Et-β-Glc(Ac)₂, -S-Et-β-Glc(Ac)₃,-S-Et-β-Glc(Ac)₄, -S-Et-α-Man(Ac)₃, -S-Et-α-Man(Ac)₄, -S-Et-β-Gal(Ac)₃,-S-Et-β-Gal(Ac)₄, -S-Et-Lac(Ac)₅, -S-Et-Lac(Ac)₆, or -S-Et-Lac(Ac)₇. 34.A method according to claim 27, wherein the carbohydrate moiety is adendrimer moiety.
 35. A method of determining the structure-functionrelationships of chemically modified mutant proteins comprising:providing first and second chemically modified mutant proteins accordingto claim 1, wherein at least one different cysteine residue in thesecond chemically modified mutant enzyme is modified by reacting saidcysteine residue with a glycosylated thiosulfonate; evaluating afunctional characteristic of the first and second chemically modifiedmutant proteins; and correlating the functional characteristic of thefirst and second chemically modified mutant proteins with the structuresof the first and second chemically modified mutant proteins. 36 A methodaccording to claim 35, wherein the protein is an enzyme.
 37. A methodaccording to claim 36, wherein the enzyme is a protease.
 38. A methodaccording to claim 37, wherein the protease is a Bacillus lentussubtilisin.
 39. A method according to claim 35, wherein the residueother than cysteine is an amino acid selected from the group consistingof asparagine, leucine, and serine.
 40. A method according to claim 35,wherein the residue other than cysteine is in a substrate bindingsubsite of the protein.
 41. A method according to claim 35, wherein theglycosylated thiosulfonate comprises a thiol side chain comprising-S-β-Glc, -S-Et-β-Gal, -S-Et-β-Glc, -S-Et-α-Glc, -S-Et-α-Man, -S-Et-Lac,-S-β-Glc(Ac)₂, -S-β-Glc(Ac)₃, -S-β-Glc(Ac)₄, -S-Et-α-Glc(Ac)₂,-S-Et-α-Glc(Ac)₃, -S-Et-α-Glc(Ac)₄, -S-Et-β-Glc(Ac)₂, -S-Et-β-Glc(Ac)₃,-S-Et-β-Glc(Ac)₄, -S-Et-α-Man(Ac)₃, -S-Et-α-Man(Ac)₄, -S-Et-β-Gal(Ac)₃,-S-Et-β-Gal(Ac)₄, -S-Et-Lac(Ac)₅, -S-Et-Lac(Ac)₆, or -S-Et-Lac(Ac)₇. 42.A method according to claim 35, wherein the carbohydrate moiety is adendrimer moiety.